Nucleic acid and corresponding protein entitled 161P5C5 useful in treatment and detection of cancer

ABSTRACT

A novel gene (designated 161P5C5) and its encoded protein, and variants thereof, are described wherein 161P5C5 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 161P5C5 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 161P5C5 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 161P5C5 can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/442,031 filed May26, 2006, now allowed, which is a continuation of U.S. Ser. No.10/120,885 filed Apr. 9, 2002, which claims benefit under 35 U.S.C.§119(e) to U.S. Ser. Nos. 60/286,630 filed Apr. 25, 2001 and 60/283,112filed Apr. 10, 2001. The contents of these applications are incorporatedherein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEb

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference inits entirety for all purposes. The sequence listing is identified on theelectronically filed text file as follows:

File Name Date of Creation Size (bytes) 511582006802Seqlist.txt December23, 2009 61,717 bytes

TECHNICAL FIELD

The invention described herein relates to a gene and its encodedprotein, termed 161P5C5, expressed in certain cancers, and to diagnosticand therapeutic methods and compositions useful in the management ofcancers that express 161P5C5.

BACKGROUND ART

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein, et al., Nat. Med. (1997)3:402). More recently identified prostate cancer markers include PCTA-1(Su, et al., Proc. Natl. Acad. Sci. USA (1996) 93:7252),prostate-specific membrane (PSM) antigen (Pinto, et al., Clin Cancer Res(1996) 9:1445-1451), STEAP (Hubert, et al., Proc. Natl. Acad. Sci. USA(1999) 96:14523-14528) and prostate stem cell antigen (PSCA) (Reiter, etal., Proc. Natl. Acad. Sci. USA (1998) 95:1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or ureter. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and 8 per 100,000 in women. The historic male/femaleratio of 3:1 may be decreasing related to smoking patterns in women.There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800in men and 3,900 in women). Bladder cancer incidence and mortalitystrongly increase with age and will be an increasing problem as thepopulation becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andintravesical chemotherapy or immunotherapy. The multifocal and recurrentnature of bladder cancer points out the limitations of TUR. Mostmuscle-invasive cancers are not cured by TUR alone. Radical cystectomyand urinary diversion is the most effective means to eliminate thecancer but carry an undeniable impact on urinary and sexual function.There continues to be a significant need for treatment modalities thatare beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(−2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation, is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980s, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for pancreatic cancer.

DISCLOSURE OF THE INVENTION

The present invention relates to a gene, designated 161P5C5, that hasnow been found to be over-expressed in the cancer(s) listed in Table I.Northern blot expression analysis of 161P5C5 gene expression in normaltissues shows a restricted expression pattern in adult tissues. Thenucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of161P5C5 are provided. The tissue-related profile of 161P5C5 in normaladult tissues, combined with the over-expression observed in the tissueslisted in Table I, shows that 161P5C5 is aberrantly over-expressed in atleast some cancers, and thus serves as a useful diagnostic,prophylactic, prognostic, and/or therapeutic target for cancers of thetissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 161P5C5 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding161P5C5-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 71or more than 71 contiguous amino acids of a 161P5C5-related protein, aswell as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, andrelated molecules, polynucleotides or oligonucleotides complementary orhaving at least a 90% homology to the 161P5C5 genes or mRNA sequences orparts thereof, and polynucleotides or oligonucleotides that hybridize tothe 161P5C5 genes, mRNAs, or to 161P5C5-encoding polynucleotides. Alsoprovided are means for isolating cDNAs and the genes encoding 161P5C5.Recombinant DNA molecules containing 161P5C5 polynucleotides, cellstransformed or transduced with such molecules, and host-vector systemsfor the expression of 161P5C5 gene products are also provided. Theinvention further provides antibodies that bind to 161P5C5 proteins andpolypeptide fragments thereof, including polyclonal and monoclonalantibodies, murine and other mammalian antibodies, chimeric antibodies,humanized and fully human antibodies, and antibodies labeled with adetectable marker or therapeutic agent. In certain embodiments there isa proviso that the entire nucleic acid sequence of FIG. 2 is not encodedand/or the entire amino acid sequence of FIG. 2 is not prepared. Incertain embodiments, the entire nucleic acid sequence of FIG. 2 isencoded and/or the entire amino acid sequence of FIG. 2 is prepared,either of which are in respective human unit dose forms.

The invention further provides methods for detecting the presence andstatus of 161P5C5 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 161P5C5.A typical embodiment of this invention provides methods for monitoring161P5C5 gene products in a tissue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 161P5C5such as cancers of tissues listed in Table I, including therapies aimedat inhibiting the transcription, translation, processing or function of161P5C5 as well as cancer vaccines. In one aspect, the inventionprovides compositions, and methods comprising them, for treating acancer that expresses 161P5C5 in a human subject wherein the compositioncomprises a carrier suitable for human use and a human unit dose of oneor more than one agent that inhibits the production or function of161P5C5. Preferably, the carrier is a uniquely human carrier. In anotheraspect of the invention, the agent is a moiety that is immunoreactivewith 161P5C5 protein. Non-limiting examples of such moieties include,but are not limited to, antibodies (such as single chain, monoclonal,polyclonal, humanized, chimeric, or human antibodies), functionalequivalents thereof (whether naturally occurring or synthetic), andcombinations thereof. The antibodies can be conjugated to a diagnosticor therapeutic moiety. In another aspect, the agent is a small moleculeas defined herein.

In another aspect, the agent comprises one or more than one peptidewhich comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLAclass I molecule in a human to elicit a CTL response to 161P5C5 and/orone or more than one peptide which comprises a helper T lymphocyte (HTL)epitope which binds an HLA class II molecule in a human to elicit an HTLresponse. The peptides of the invention may be on the same or on one ormore separate polypeptide molecules. In a further aspect of theinvention, the agent comprises one or more than one nucleic acidmolecule that expresses one or more than one of the CTL or HTL responsestimulating peptides as described above. In yet another aspect of theinvention, the one or more than one nucleic acid molecule may express amoiety that is immunologically reactive with 161P5C5 as described above.The one or more than one nucleic acid molecule may also be, or encodes,a molecule that inhibits production of 161P5C5. Non-limiting examples ofsuch molecules include, but are not limited to, those complementary to anucleotide sequence essential for production of 161P5C5 (e.g. antisensesequences or molecules that form a triple helix with a nucleotide doublehelix essential for 161P5C5 production) or a ribozyme effective to lyse161P5C5 mRNA.

Note: To determine the starting position of any peptide set forth inTables V-XVIII and XXII to LI (collectively HLA Peptide Tables)respective to its parental protein, e.g., variant 1, variant 2, etc.,reference is made to three factors: the particular variant, the lengthof the peptide in an HLA Peptide Table, and the Search Peptides in TableLII. Generally, a unique Search Peptide is used to obtain HLA peptidesof a partiular for a particular variant. The position of each SearchPeptide relative to its respective parent molecule is listed in TableLII. Accordingly if a Search Peptide begins at position “X”, one mustadd the value “X-1” to each position in Tables V-XVIII and XXII to LI toobtain the actual position of the HLA peptides in their parentalmolecule. For example if a particular Search Peptide begins at position150 of is parental molecule, one must add 150−1, i.e., 149 to each HLApeptide amino acid position to calculate the position of that amino acidin the parent molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.A. The 161P5C5 SSH sequence of 95 nucleotides. B. The 163P3C6 SSHsequence of 467 nucleotides.

FIG. 2A. The cDNA and amino acid sequence of 161P5C5 v.1. The startmethionine is underlined. The open reading frame extends from nucleicacid 1035-1250 including the stop codon.

FIG. 2B. The cDNA and amino acid sequence of 161P5C5 v.2. The startmethionine is underlined. The open reading frame extends from nucleicacid 1035-1250 including the stop codon.

FIG. 2C. The cDNA and amino acid sequence of 161P5C5 v.3. The startmethionine is underlined. The open reading frame extends from nucleicacid 1035-1250 including the stop codon.

FIG. 2D. The cDNA and amino acid sequence of 161P5C5 v.4. The startmethionine is underlined. The open reading frame extends from nucleicacid 1035-1250 including the stop codon.

FIG. 2E. The cDNA and amino acid sequence of 161P5C5 v.5. The startmethionine is underlined. The open reading frame extends from nucleicacid 1035-1250 including the stop codon.

FIG. 2F. The cDNA and amino acid sequence of 161P5C5 v.6. The startmethionine is underlined. The open reading frame extends from nucleicacid 1035-1250 including the stop codon.

FIG. 2G. The cDNA and amino acid sequence of 161P5C5 v.7. The startmethionine is underlined. The open reading frame extends from nucleicacid 896-1111 including the stop codon.

As used herein, a reference to 161P5C5 includes all variants thereof,including those shown in FIG. 10, unless a variant is specified.

FIG. 3A. Amino acid sequence of 161P5C5 v.1. The 161P5C5 v.1 protein has71 amino acids.

FIG. 3B. Amino acid sequence of 161P5C5 v.2. The 161P5C5 v.2 protein has71 amino acids.

FIG. 3C. Amino acid sequence of 161P5C5 v.3 . The 161P5C5 v.3 proteinhas 71 amino acids.

As used herein, a reference to 161P5C5 includes all variants thereof,including those shown in FIG. 11, unless a variant is specified.

FIG. 4A. Nucleic acid alignment of 161P5C5 variants. SNPs areunderlined.

FIG. 4B. Amino Acid Alignment of 161P5C5 variants. SNPs are underlined.

FIG. 4C. Comparison of 161P5C5-V.1 with known genes: alignment withBindin Fertilization Specific Protein.

FIG. 4D. Comparison of 161P5C5-V.1 with known genes: alignment withprotoporphyrinogen oxidase.

FIG. 5. Hydrophilicity amino acid profile of 161P5C5 variant 1,determined by computer algorithm sequence analysis using the method ofHopp and Woods (Hopp, T. P., Woods, K. R., Proc. Natl. Acad. Sci. USA(1981) 78:3824-3828) accessed on the Protscale website located on theWorld Wide Web through the ExPasy molecular biology server.

FIG. 6. Hydropathicity amino acid profile of 161P5C5 variant 1,determined by computer algorithm sequence analysis using the method ofKyte and Doolittle (Kyte, J., Doolittle, R. F., J. Mol. Biol. (1982)157:105-132) accessed on the ProtScale website located on the World WideWeb through the ExPasy molecular biology server.

FIG. 7. Percent accessible residues amino acid profile of 161P5C5variant 1, determined by computer algorithm sequence analysis using themethod of Janin (Janin, J., Nature (1979) 277:491-492) accessed on theProtScale website located on the World Wide Web through the ExPasymolecular biology server.

FIG. 8. Average flexibility amino acid profile of 161P5C5 variant 1,determined by computer algorithm sequence analysis using the method ofBhaskaran and Ponnuswamy (Bhaskaran, R., and Ponnuswamy, P. K., Int. J.Pept. Protein Res. (1988) 32:242-255) accessed on the ProtScale websitelocated on the World Wide Web through the ExPasy molecular biologyserver.

FIG. 9. Beta-turn amino acid profile of 161P5C5 variant 1, determined bycomputer algorithm sequence analysis using the method of Deleage andRoux (Deleage, G., Roux, B., Protein Engineering (1987) 1:289-294)accessed on the ProtScale website located on the World Wide Web throughthe ExPasy molecular biology server.

FIG. 10. Schematic display of nucleotide variants of 161P5C5. Variant161P5C5 v.2 through 161P5C5 v.6 are variants with single nucleotidevariations. Variant 161P5C5 v.7 is a splice variant. The black boxesshow the same sequence as 161P5C5 v.1. Numbers in “( ) ” underneath thebox correspond to those of 161P5C5 v.1. SNPs are indicated above thebox.

FIG. 11. Schematic display of protein variants of 161P5C5. Nucleotidevariants 161P5C5 v.1, v.2 and v.3 in FIG. 10 code for protein variants161P5C5 v.1, v.2 and v.3. Nucleotide variants 161P5C5 v.4 through v.7 inFIG. 10 code for the same protein as variant 161P5C5 v.1. The blackboxes show the same sequence as 161P5C5 v.1. Numbers correspond to thoseof 161P5C5 v.1. Single amino acid differences are indicated above thebox.

FIG. 12. Exon compositions of transcript variants of 161P5C5. Variant161P5C5 v.7 is a splice variant that spliced out exon 2 along withintrons. However, they code for the same protein. Numbers in “( )”underneath the box correspond to those of 161P5C5 v.1. Black boxes showthe same sequence as 161P5C5 v.1. Length of introns are notproportional.

FIG. 13. Secondary structure prediction for 161P5C5. (SEQ ID NO:56). Thesecondary structure of 161P5C5 variant 1 was predicted using theHNN—Hierarchical Neural Network method (Guermeur, 1997), accessed fromthe ExPasy molecular biology server. This method predicts the presenceand location of alpha helices, extended strands, and random coils fromthe primary protein sequence. The percent of the protein in a givensecondary structure is also listed.

FIG. 14. Expression of 161P5C5 by RT-PCR. First strand cDNA was preparedfrom vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas,colon and stomach), bladder cancer pool, kidney cancer pool, lung cancerpool, ovary cancer pool, breast cancer pool, and cancer metastasis pool.Normalization was performed by PCR using primers to actin and GAPDH.Semi-quantitative PCR, using primers to 161P5C5, was performed at 26 and30 cycles of amplification. Results show strong expression of 161P5C5 inbladder cancer pool, kidney cancer pool, ovary cancer pool, breastcancer pool, and cancer metastasis pool. Expression of 161P5C5 was alsodetected in lung cancer pool, but not in vital pool 1, and vital pool 2.

FIG. 15. Expression of 161P5C5 in normal tissues. Two multiple tissuenorthern blots (Clontech) both with 2 μg of mRNA/lane were probed withthe 161P5C5 sequence. Size standards in kilobases (kb) are indicated onthe side. Results show absence of expression of 161P5C5 in all 16 normaltissues tested.

FIG. 16. Expression of 161P5C5 in bladder cancer patient tissues. RNAwas extracted from normal bladder (NB), bladder cancer cell lines (CL;UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) and normaladjacent tissue (N). Northern blots with 10 μg of total RNA were probedwith the 161P5C5 SSH sequence. Size standards in kilobases are indicatedon the side. Results show strong expression of 161P5C5 in patientbladder cancer tissues but not in normal bladder nor in the bladdercancer cell lines.

FIG. 17. Expression of 161P5C5 in kidney cancer patient tissues. RNA wasextracted from normal kidney (NK), kidney cancer patient tumors (T) andtheir normal adjacent tissues (N). Northern blots with 10 μs of totalRNA were probed with the 161P5C5 SSH sequence. Size standards inkilobases are on the side. Results show strong expression of 161P5C5 inpatient kidney cancer tissues, but not in normal kidney.

FIG. 18. Expression of 161P5C5 in ovary cancer patient tissues. RNA wasextracted from ovary and cervical cancer cell lines (CL), normal ovary(N), and ovary cancer patient tumors (T). Northern blots with 10 μg oftotal RNA were probed with the 161P5C5 SSH sequence. Size standards inkilobases are on the side. Results show strong expression of 161P5C5 inpatient ovary cancer tissues, but not in normal ovary nor in the ovaryand cervical cancer cell lines.

MODES OF CARRYING OUT THE INVENTION

Definitions:

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd.edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. As appropriate, procedures involving the use of commerciallyavailable kits and reagents are generally carried out in accordance withmanufacturer defined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 161P5C5 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 161P5C5. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g.,a 161P5C5-related protein). For example, an analog of a 161P5C5 proteincan be specifically bound by an antibody or T cell that specificallybinds to 161P5C5.

The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-161P5C5antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-161P5C5 antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-161P5C5 antibodycompositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to maytansinoids, yttrium,bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol,ethidium bromide, mitomycin, etoposide, teniposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin,diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,sapaonaria officinalis inhibitor, and glucocorticoid and otherchemotherapeutic agents, as well as radioisotopes such as At²¹¹, I¹³¹,I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes ofLu. Antibodies may also be conjugated to an anti-cancer pro-drugactivating enzyme capable of converting the pro-drug to its active form.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,Immunology, 8th Ed., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37° C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55° C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 161P5C5 genes orthat encode polypeptides other than 161P5C5 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated 161P5C5 polynucleotide. A protein issaid to be “isolated,” for example, when physical, mechanical orchemical methods are employed to remove the 161P5C5 proteins fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 161P5C5 protein. Alternatively, an isolated proteincan be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” meanprostate cancers that have spread to regional lymph nodes or to distantsites, and are meant to include stage D disease under the AUA system andstage T×N×M+ under the TNM system. As is the case with locally advancedprostate cancer, surgery is generally not indicated for patients withmetastatic disease, and hormonal (androgen ablation) therapy is apreferred treatment modality. Patients with metastatic prostate cancereventually develop an androgen-refractory state within 12 to 18 monthsof treatment initiation. Approximately half of these androgen-refractorypatients die within 6 months after developing that status. The mostcommon site for prostate cancer metastasis is bone. Prostate cancer bonemetastases are often osteoblastic rather than osteolytic (i.e.,resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the antibodiescomprising the population are identical except for possible naturallyoccurring mutations that are present in minor amounts.

A “motif”, as in biological motif of a 161P5C5-related protein, refersto any pattern of amino acids forming part of the primary sequence of aprotein, that is associated with a particular function (e.g.protein-protein interaction, protein-DNA interaction, etc.) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 2, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class I molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. In another embodiment, for example, the primary anchorresidues of a peptide that will bind an HLA class II molecule are spacedrelative to each other, rather than to the termini of a peptide, wherethe peptide is generally of at least 9 amino acids in length. Theprimary anchor positions for each motif and supermotif are set forth inTable IV. For example, analog peptides can be created by altering thepresence or absence of particular residues in the primary and/orsecondary anchor positions shown in Table IV. Such analogs are used tomodulate the binding affinity and/or population coverage of a peptidecomprising a particular HLA motif or supermotif.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind orinteract with 161P5C5, ligands including hormones, neuropeptides,chemokines, odorants, phospholipids, and functional equivalents thereofthat bind and preferably inhibit 161P5C5 protein function. Suchnon-limiting small molecules preferably have a molecular weight of lessthan about 10 kDa, more preferably below about 9, about 8, about 7,about 6, about 5 or about 4 kDa. In certain embodiments, small moleculesphysically associate with, or bind, 161P5C5 protein; are not found innaturally occurring metabolic pathways; and/or are more soluble inaqueous than non-aqueous solutions.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel,et al., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% FicoII/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook, et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

An HLA “supermotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles.

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; fulleradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g., the 161P5C5 protein shown in FIG. 2 or FIG. 3.An analog is an example of a variant protein. Splice isoforms and singlenucleotides polymorphisms (SNPs) are further examples of variants.

The “161P5C5-related proteins” of the invention include thosespecifically identified herein, as well as allelic variants,conservative substitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 161P5C5 proteins orfragments thereof, as well as fusion proteins of a 161P5C5 protein and aheterologous polypeptide are also included. Such 161P5C5 proteins arecollectively referred to as the 161P5C5-related proteins, the proteinsof the invention, or 161P5C5. The term “161P5C5-related protein” refersto a polypeptide fragment or a 161P5C5 protein sequence of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, or 71 or more amino acids.

161P5C5 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a 161P5C5 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a 161P5C5-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a 161P5C5 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toa 161P5C5 gene, mRNA, or to a 161P5C5 encoding polynucleotide(collectively, “161P5C5 polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 161P5C5 polynucleotide include: a 161P5C5polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 161P5C5 as shown in FIG. 2 wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 161P5C5 nucleotides comprise, without limitation:

(I) a polynucleotide comprising, consisting essentially of, orconsisting of a sequence as shown in FIG. 2, wherein T can also be U;

(II) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2A, from nucleotide residuenumber 1035 through nucleotide residue number 1250, including the stopcodon, wherein T can also be U;

(III) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2B, from nucleotide residuenumber 1035 through nucleotide residue number 1250, including the stopcodon, wherein T can also be U;

(IV) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2C, from nucleotide residuenumber 1035 through nucleotide residue number 1250, including the a stopcodon, wherein T can also be U;

(V) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2D, from nucleotide residuenumber 1035 through nucleotide residue number 1250, including the stopcodon, wherein T can also be U;

(VI) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2E, from nucleotide residuenumber 1035 through nucleotide residue number 1250, including the stopcodon, wherein T can also be U;

(VII) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2F, from nucleotide residuenumber 1035 through nucleotide residue number 1250, including the stopcodon, wherein T can also be U;

(VIII) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2G, from nucleotide residuenumber 896 through nucleotide residue number 1111, including the stopcodon, wherein T can also be U;

(IX) a polynucleotide that encodes a 161P5C5-related protein that is atleast 90% homologous to an entire amino acid sequence shown in FIG.2A-G;

(X) a polynucleotide that encodes a 161P5C5-related protein that is atleast 90% identical to an entire amino acid sequence shown in FIG. 2A-G;

(XI) a polynucleotide that encodes at least one peptide set forth inTables V-XVIII and XXII-LI;

(XII) a polynucleotide that encodes a peptide region of at least 5 aminoacids of a peptide of FIG. 3A in any whole number increment up to 71that includes an amino acid position having a value greater than 0.5 inthe Hydrophilicity profile of FIG. 5;

(XIII) a polynucleotide that encodes a peptide region of at least 5amino acids of a peptide of FIG. 3A in any whole number increment up to71 that includes an amino acid position having a value less than 0.5 inthe Hydropathicity profile of FIG. 6;

(XIV) a polynucleotide that encodes a peptide region of at least 5 aminoacids of a peptide of FIG. 3A in any whole number increment up to 71that includes an amino acid position having a value greater than 0.5 inthe Percent Accessible Residues profile of FIG. 7;

(XV) a polynucleotide that encodes a peptide region of at least 5 aminoacids of a peptide of FIG. 3A in any whole number increment up to 71that includes an amino acid position having a value greater than 0.5 inthe Average Flexibility profile of FIG. 8;

(XVI) a polynucleotide that encodes a peptide region of at least 5 aminoacids of a peptide of FIG. 3A in any whole number increment up to 71that includes an amino acid position having a value greater than 0.5 inthe Beta-turn profile of FIG. 9;

(XVII) a polynucleotide that is fully complementary to a polynucleotideof any one of (I)-(XVI).

(XVIII) a polynucleotide that encodes a 161P5C5-related protein whosesequence is encoded by the cDNAs contained in the plasmid deposited withAmerican Type Culture Collection (ATCC; 10801 University Blvd.,Manassas, VA 20110-2209 USA) as Accession No. ATCC-PTA-4184 on Mar. 28,2002; and

(XIX) a peptide that is encoded by any of (I)-(XVIII);

(XX) a polynucleotide of any of (I)-(XVIII) or peptide of (XIX) togetherwith a pharmaceutical excipient and/or in a human unit dose form.

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 161P5C5polynucleotides that encode specific portions of 161P5C5 mRNA sequences(and those which are complementary to such sequences) such as those thatencode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 71 contiguousamino acids of 161P5C5.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 161P5C5protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 161P5C5 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 20 to about amino acid30 of the 161P5C5 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 30 to about amino acid 40 of the 161P5C5protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 40 to about amino acid 50 of the 161P5C5 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 50 to about amino acid60 of the 161P5C5 protein shown in FIG. 2 or FIG. 3, or polynucleotidesencoding about amino acid 60 to about amino acid 70 or amino acid 71 ofthe 161P5C5 protein shown in FIG. 2 or FIG. 3. Accordinglypolynucleotides encoding portions of the amino acid sequence (of about10 amino acids), of amino acids 1 through the carboxyl terminal aminoacid of the 161P5C5 protein are embodiments of the invention. Wherein itis understood that each particular amino acid position discloses thatposition plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a 161P5C5 proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the 161P5C5 protein “or variant”shown in FIG. 2 or FIG. 3 can be generated by a variety of techniqueswell known in the art. These polynucleotide fragments can include anyportion of the 161P5C5 sequence as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed hereininclude 161P5C5 polynucleotide fragments encoding one or more of thebiological motifs contained within a 161P5C5 protein “or variant”sequence, including one or more of the motif-bearing subsequences of a161P5C5 protein “or variant” set forth in Tables V-XVIII and XXII-LI. Inanother embodiment, typical polynucleotide fragments of the inventionencode one or more of the regions of 161P5C5 protein or variant thatexhibit homology to a known molecule. In another embodiment of theinvention, typical polynucleotide fragments can encode one or more ofthe 161P5C5 protein or variant N-glycosylation sites, cAMP andcGMP-dependent protein kinase phosphorylation sites, casein kinase IIphosphorylation sites or N-myristoylation site and amidation sites.

Note that to determine the starting position of any peptide set forth inTables V-XVIII and Tables XXII-LI (collectively HLA Peptide Tables)respective to its parental protein, e.g., variant 1, variant 2, etc.,reference is made to three factors: the particular variant, the lengthof the peptide in an HLA Peptide Table, and the Search Peptides listedin Table LLII. Generally, a unique Search Peptide is used to obtain HLApeptides for a particular variant. The position of each Search Peptiderelative to its respective parent molecule is listed in Table LLII.Accordingly if a Search Peptide begins at position “X”, one must add thevalue “X-1” to each position in Tables V-XVIII and Tables XXII-LLI toobtain the actual position of the HLA peptides in their parentalmolecule. For example if a particular Search Peptide begins at position150 of its parental molecule, one must add 150−1, i.e., 149 to each HLApeptide amino acid position to calculate the position of that amino acidin the parent molecule.

One embodiment of the invention comprises an HLA peptide, that occurs atleast twice in Tables V-XVIII and XXII to LI collectively, or anoligonucleotide that encodes the HLA peptide. Another embodiment of theinvention comprises an HLA peptide that occurs at least once in TablesV-XVIII and at least once in tables XXII to LI, or an oligonucleotidethat encodes the HLA peptide.

Another embodiment of the invention is antibody epitopes which comprisea peptide regions, or an oligonucleotide encoding the peptide region,that has one two, three, four, or five of the following characteristics:

-   -   i) a peptide region of at least 5 amino acids of a particular        peptide of FIG. 3, in any whole number increment up to the full        length of that protein in FIG. 3, that includes an amino acid        position having a value equal to or greater than 0.5, 0.6, 0.7,        0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity        profile of FIG. 5;    -   ii) a peptide region of at least 5 amino acids of a particular        peptide of FIG. 3, in any whole number increment up to the full        length of that protein in FIG. 3, that includes an amino acid        position having a value equal to or less than 0.5, 0.4, 0.3,        0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity        profile of FIG. 6;    -   iii) a peptide region of at least 5 amino acids of a particular        peptide of FIG. 3, in any whole number increment up to the full        length of that protein in FIG. 3, that includes an amino acid        position having a value equal to or greater than 0.5, 0.6, 0.7,        0.8, 0.9, or having a value equal to 1.0, in the Percent        Accessible Residues profile of FIG. 7;    -   iv) a peptide region of at least 5 amino acids of a particular        peptide of FIG. 3, in any whole number increment up to the full        length of that protein in FIG. 3, that includes an amino acid        position having a value equal to or greater than 0.5, 0.6, 0.7,        0.8, 0.9, or having a value equal to 1.0, in the Average        Flexibility profile of FIG. 8; or    -   v) a peptide region of at least 5 amino acids of a particular        peptide of FIG. 3, in any whole number increment up to the full        length of that protein in FIG. 3, that includes an amino acid        position having a value equal to or greater than 0.5, 0.6, 0.7,        0.8, 0.9, or having a value equal to 1.0, in the Beta-turn        profile of FIG. 9.

Uses of 161P5C5 Polynucleotides

Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 161P5C5 gene maps to the chromosomallocation set forth in the Example entitled “Chromosomal Mapping of161P5C5.” For example, because the 161P5C5 gene maps to this chromosome,polynucleotides that encode different regions of the 161P5C5 proteinsare used to characterize cytogenetic abnormalities of this chromosomallocale, such as abnormalities that are identified as being associatedwith various cancers. In certain genes, a variety of chromosomalabnormalities including rearrangements have been identified as frequentcytogenetic abnormalities in a number of different cancers (see, e.g.,Krajinovic, et al., Mutat. Res. (1998) 382:81-83; Johansson, et al.,Blood (1995) 86:3905-3914 and Finger, et al., P.N.A.S. (1988)85:9158-9162). Thus, polynucleotides encoding specific regions of the161P5C5 proteins provide new tools that can be used to delineate, withgreater precision than previously possible, cytogenetic abnormalities inthe chromosomal region that encodes 161P5C5 that may contribute to themalignant phenotype. In this context, these polynucleotides satisfy aneed in the art for expanding the sensitivity of chromosomal screeningin order to identify more subtle and less common chromosomalabnormalities (see, e.g., Evans, et al., Am. J. Obstet. Gynecol (1994)171:1055-1057).

Furthermore, as 161P5C5 was shown to be highly expressed in bladder andother cancers, 161P5C5 polynucleotides are used in methods assessing thestatus of 161P5C5 gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the 161P5C5proteins are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 161P5C5 gene, suchas regions containing one or more motifs. Exemplary assays include bothRT-PCR assays as well as single-strand conformation polymorphism (SSCP)analysis (see, e.g., Marrogi, et al., J. Cutan. Pathol. (1999)26:369-378, both of which utilize polynucleotides encoding specificregions of a protein to examine these regions within the protein.

Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 161P5C5. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 161P5C5 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,161P5C5. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis (1988)1:1-5. The 161P5C5 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See, e.g., Iyer, R. P., et al., J. Org. Chem. (1990)55:4693-4698; and Iyer, R. P., et al., J. Am. Chem. Soc. (1990)112:1253-1254. Additional 161P5C5 antisense oligonucleotides of thepresent invention include morpholino antisense oligonucleotides known inthe art (see, e.g., Partridge, et al., Antisense & Nucleic Acid DrugDevelopment (1996) 6:169-175).

The 161P5C5 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of a161P5C5 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 161P5C5 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 161P5C5 antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 161P5C5 mRNA. Optionally, 161P5C5antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 161P5C5. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 161P5C5 expression, see, e.g.,Couture, L. A., and Stinchcomb, D. T., Trends Genet (1996) 12:510-515.

Primers and Primer Pairs

Further specific embodiments of this nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 161P5C5 polynucleotide in a sample and as ameans for detecting a cell expressing a 161P5C5 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 161P5C5 cDNA sequence shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 161P5C5 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 161P5C5 mRNA.

The 161P5C5 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 161P5C5 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 161P5C5 polypeptides; as tools formodulating or inhibiting the expression of the 161P5C5 gene(s) and/ortranslation of the 161P5C5 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described hereinto identify and isolate a 161P5C5 or 161P5C5 related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

Isolation of 161P5C5-Encoding Nucleic Acid Molecules

The 161P5C5 cDNA sequences described herein enable the isolation ofother polynucleotides encoding 161P5C5 gene product(s), as well as theisolation of polynucleotides encoding 161P5C5 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms of a161P5C5 gene product as well as polynucleotides that encode analogs of161P5C5-related proteins. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding a 161P5C5 gene are wellknown (see, for example, Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology, Ausubel, et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 161P5C5gene cDNAs can be identified by probing with a labeled 161P5C5 cDNA or afragment thereof. For example, in one embodiment, a 161P5C5 cDNA (e.g.,FIG. 2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a 161P5C5gene. A 161P5C5 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 161P5C5 DNAprobes or primers.

Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga 161P5C5 polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook, et al., 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 161P5C5 polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of 161P5C5or a fragment, analog or homolog thereof can be used to generate 161P5C5proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of161P5C5 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller, et al., MCB (1991) 11:1785). Using theseexpression vectors, 161P5C5 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 161P5C5 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 161P5C5 and 161P5C5 mutations or analogs.

Recombinant human 161P5C5 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 161P5C5-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 161P5C5 or fragment,analog or homolog thereof, a 161P5C5-related protein is expressed in the293T cells, and the recombinant 161P5C5 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-161P5C5 antibodies). In another embodiment, a 161P5C5 codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish 161P5C5 expressing cell lines. Various otherexpression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to a161P5C5 coding sequence can be used for the generation of a secretedform of recombinant 161P5C5 protein.

As discussed herein, redundancy in the genetic code permits variation in161P5C5 gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol. (1989)9:5073-5080. Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak,PNAS (1995) 92:2662-2666, and Kozak, NAR (1987) 15:8125-8148).

161P5C5-Related Proteins

Another aspect of the present invention provides 161P5C5-relatedproteins. Specific embodiments of 161P5C5 proteins comprise apolypeptide having all or part of the amino acid sequence of human161P5C5 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of161P5C5 proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 161P5C5 shown in FIG. 2or FIG. 3.

In general, naturally occurring allelic variants of human 161P5C5 sharea high degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of a 161P5C5 protein containconservative amino acid substitutions within the 161P5C5 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 161P5C5. One class of 161P5C5allelic variants are proteins that share a high degree of homology withat least a small region of a particular 161P5C5 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 conservative substitutions. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g., Table III herein; pages 13-15, Biochemistry,2^(nd) ED. Lubert Stryer ed. (Stanford University); Henikoff, et al.,PNAS (1992) 89:10915-10919; Lei, et al., J Biol Chem (1995)270:11882-11886).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 161P5C5 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.161P5C5 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter, et al., Nucl. Acids Res. (1986)13:4331; Zoller, et al., Nucl. Acids Res. (1987) 10:6487), cassettemutagenesis (Wells, et al., Gene (1985) 34:315), restriction selectionmutagenesis (Wells, et al., Philos. Trans. R. Soc. London SerA (1986)317:415) or other known techniques can be performed on the cloned DNA toproduce the 161P5C5 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol. (1976) 150:1). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 161P5C5 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 161P5C5 protein having an amino acid sequence of FIG.3. As used in this sentence, “cross reactive” means that an antibody orT cell that specifically binds to a 161P5C5 variant also specificallybinds to a 161P5C5 protein having an amino acid sequence set forth inFIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG.3, when it no longer contains any epitope capable of being recognized byan antibody or T cell that specifically binds to the starting 161P5C5protein. Those skilled in the art understand that antibodies thatrecognize proteins bind to epitopes of varying size, and a grouping ofthe order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair, et al., J. Immunol (2000) 165:6949-6955; Hebbes, et al., MolImmunol (1989) 26:865-873; Schwartz, et al., J Immunol (1985)135:2598-2608.

Other classes of 161P5C5-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with an amino acid sequence of FIG. 3, ora fragment thereof. Another specific class of 161P5C5 protein variantsor analogs comprise one or more of the 161P5C5 biological motifsdescribed herein or presently known in the art. Thus, encompassed by thepresent invention are analogs of 161P5C5 fragments (nucleic or aminoacid) that have altered functional (e.g., immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of a161P5C5 protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a161P5C5 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of a 161P5C5 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of a 161P5C5protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of a 161P5C5 protein shown in FIG.2 or FIG. 3, polypeptides consisting of about amino acid 30 to aboutamino acid 40 of a 161P5C5 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofa 161P5C5 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 50 to about amino acid 60 of a 161P5C5 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 toabout amino acid 70 of a 161P5C5 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 70 to about amino acid 80 ofa 161P5C5 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 80 to about amino acid 90 of a 161P5C5 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 toabout amino acid 100 of a 161P5C5 protein shown in FIG. 2 or FIG. 3,etc. throughout the entirety of a 161P5C5 amino acid sequence. Moreover,polypeptides consisting of about amino acid 1 (or 20 or 30 or 40, etc.)to about amino acid 20, (or 130, or 140 or 150, etc.) of a 161P5C5protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. Itis to be appreciated that the starting and stopping positions in thisparagraph refer to the specified position as well as that position plusor minus 5 residues.

161P5C5-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a 161P5C5-related protein. In one embodiment,nucleic acid molecules provide a means to generate defined fragments ofa 161P5C5 protein (or variants, homologs or analogs thereof).

Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 161P5C5 polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within a 161P5C5 polypeptidesequence set forth in FIG. 2 or FIG. 3. Various motifs are known in theart, and a protein can be evaluated for the presence of such motifs by anumber of publicly available Internet sites (see, e.g., the World WideWeb; Epimatrix™ and Epimer™, Brown University; and BIMAS).

Motif bearing subsequences of all 161P5C5 variant proteins are set forthand identified in Tables V-XVIII and XXII-LII.

Table XIX sets forth several frequently occurring motifs based on pfamsearches. The columns of Table XIX list (1) motif name abbreviation, (2)percent identity found amongst the different member of the motif family,(3) motif name or description and (4) most common function; locationinformation is included if the motif is relevant for location.

Polypeptides comprising one or more of the 161P5C5 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 161P5C5 motifsdiscussed above are associated with growth dysregulation and because161P5C5 is overexpressed in certain cancers (See, e.g., Table I). Caseinkinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C,for example, are enzymes known to be associated with the development ofthe malignant phenotype (see, e.g., Chen, et al., Lab Invest. (1998)78:165-174; Gaiddon, et al., Endocrinology (1995) 136:4331-4338; Hall,et al., Nucleic Acids Research (1996) 24:1119-1126; Peterziel, et al.,Oncogene (1999) 18:6322-6329 and O'Brian, Oncol. Rep. (1998) 5:305-309).Moreover, both glycosylation and myristoylation are proteinmodifications also associated with cancer and cancer progression (see,e.g., Dennis, et al., Biochem. Biophys. Acta (1999) 1473:21-34; Raju, etal., Exp. Cell Res. (1997) 235:145-154). Amidation is another proteinmodification also associated with cancer and cancer progression (see,e.g., Treston, et al., J. Natl. Cancer Inst. Monogr. (1992) 13:169-175).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables V-XVIII and XXII-LI.CTL epitopes can be determined using specific algorithms to identifypeptides within a 161P5C5 protein that are capable of optimally bindingto specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, BrownUniversity, and BIMAS) Moreover, processes for identifying peptides thathave sufficient binding affinity for HLA molecules and which arecorrelated with being immunogenic epitopes, are well known in the art,and are carried out without undue experimentation. In addition,processes for identifying peptides that are immunogenic epitopes, arewell known in the art, and are carried out without undue experimentationeither in vitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, one can substitute out a deleterious residue infavor of any other residue, such as a preferred residue as defined inTable IV; substitute a less-preferred residue with a preferred residueas defined in Table IV; or substitute an originally-occurring preferredresidue with another preferred residue as defined in Table IV.Substitutions can occur at primary anchor positions or at otherpositions in a peptide; see, e.g., Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 97/33602 to Chesnut, et al.; Sette,Immunogenetics (1999) 50:201-212; Sette, et al., J. Immunol. (2001)166:1389-1397; Sidney, et al., Hum. Immunol. (1997) 58:12-20; Kondo, etal., Immunogenetics (1997) 45:249-258; Sidney, et al., J. Immunol.(1996) 157:3480-3490; and Falk, et al., Nature (1991) 351:290-296; Hunt,et al., Science (1992) 255:1261-1263; Parker, et al., J. Immunol. (1992)149:3580-3587; Parker, et al., J. Immunol. (1994) 152:163-175); Kast, W.M., et al., J. Immunol. (1994) 152(8):3904-3912; Borras-Cuesta, et al.,Hum. Immunol. (2000) 61:266-278; Alexander, et al., J. Immunol. (2000)164:1625-1633; Alexander, et al., PMID: 7895164, UI: 95202582;O'Sullivan, et al., J. Immunol. (1991) 147:2663-2669; Alexander, et al.,Immunity (1994) 1:751-761 and Alexander, et al., Immunol. Res. (1998)18:79-92.

Related embodiments of the invention include polypeptides comprisingcombinations of the different motifs set forth in Table XX, and/or, oneor more of the predicted CTL epitopes of Tables V-XVII and XXII-XLVII,and/or, one or more of the predicted HTL epitopes of Tables XLVIII-LI,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typicallythe number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

161P5C5-related proteins are embodied in many forms, preferably inisolated form. A purified 161P5C5 protein molecule will be substantiallyfree of other proteins or molecules that impair the binding of 161P5C5to antibody, T cell or other ligand. The nature and degree of isolationand purification will depend on the intended use. Embodiments of a161P5C5-related proteins include purified 161P5C5-related proteins andfunctional, soluble 161P5C5-related proteins. In one embodiment, afunctional, soluble 161P5C5 protein or fragment thereof retains theability to be bound by antibody, T cell or other ligand.

The invention also provides 161P5C5 proteins comprising biologicallyactive fragments of a 161P5C5 amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the starting 161P5C5protein, such as the ability to elicit the generation of antibodies thatspecifically bind an epitope associated with the starting 161P5C5protein; to be bound by such antibodies; to elicit the activation of HTLor CTL; and/or, to be recognized by HTL or CTL that also specificallybind to the starting protein.

161P5C5-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or on the basis of immunogenicity. Fragmentsthat contain such structures are particularly useful in generatingsubunit-specific anti-161P5C5 antibodies, or T cells or in identifyingcellular factors that bind to 161P5C5. For example, hydrophilicityprofiles can be generated, and immunogenic peptide fragments identified,using the method of Hopp, T. P. and Woods, K. R., Proc. Natl. Acad. Sci.U.S.A. (1981) 78:3824-3828. Hydropathicity profiles can be generated,and immunogenic peptide fragments identified, using the method of Kyte,J. and Doolittle, R. F., J. Mol. Biol. (1982) 157:105-132. Percent (%)Accessible Residues profiles can be generated, and immunogenic peptidefragments identified, using the method of Janin, J., Nature (1979)277:491-492. Average Flexibility profiles can be generated, andimmunogenic peptide fragments identified, using the method of Bhaskaran,R., and Ponnuswamy, P. K., Int. J. Pept. Protein Res. (1988) 32:242-255.Beta-turn profiles can be generated, and immunogenic peptide fragmentsidentified, using the method of Deleage, G., and Roux, B., ProteinEngineering (1987) 1:289-294.

CTL epitopes can be determined using specific algorithms to identifypeptides within a 161P5C5 protein that are capable of optimally bindingto specified HLA alleles (e.g., by using the SYFPEITHI site on the WorldWide Web; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, BrownUniversity; and BIMAS. Illustrating this, peptide epitopes from 161P5C5that are presented in the context of human MHC Class I molecules, e.g.,HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., TablesV-XVIII, XXII-LI). Specifically, the complete amino acid sequence of the161P5C5 protein and relevant portions of other variants, i.e., for HLAClass I predictions 9 flanking residues on either side of a pointmutation, and for HLA Class II predictions 14 flanking residues oneither side of a point mutation, were entered into the HLA Peptide MotifSearch algorithm found in the Bioinformatics and Molecular AnalysisSection (BIMAS) web site; and the site SYFPEITHI was used.

The HLA peptide motif search algorithm was developed by Dr. Ken Parkerbased on binding of specific peptide sequences in the groove of HLAClass I molecules, in particular HLA-A2 (see, e.g., Falk, et al., Nature(1991) 351:290-296; Hunt, et al., Science (1992) 255:1261-1263; Parker,et al., J. Immunol. (1992) 149:3580-3587; Parker, et al., J. Immunol.(1994) 152:163-175). This algorithm allows location and ranking of8-mer, 9-mer, and 10-mer peptides from a complete protein sequence forpredicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for Class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker, et al., J. Immunol. (1992)149:3580-3587). Selected results of 161P5C5 predicted binding peptidesare shown in Tables V-XVIII and XXII-LI herein. In Tables V-XVIII andXXII-LI, selected candidates, 9-mers, 10-mers, and 15-mers for eachfamily member are shown along with their location, the amino acidsequence of each specific peptide, and an estimated binding score. Thebinding score corresponds to the estimated half time of dissociation ofcomplexes containing the peptide at 37° C. at pH 6.5. Peptides with thehighest binding score are predicted to be the most tightly bound to HLAClass I on the cell surface for the greatest period of time and thusrepresent the best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue, et al., Prostate (1997) 30:73-78 and Peshwa, etal., Prostate (1998) 36:129-138). Immunogenicity of specific peptidescan be evaluated in vitro by stimulation of CD8+cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV (or determined using World Wide Web site, orBIMAS) are to be “applied” to a 161P5C5 protein in accordance with theinvention. As used in this context “applied” means that a 161P5C5protein is evaluated, e.g., visually or by computer-based patternsfinding methods, as appreciated by those of skill in the relevant art.Every subsequence of a 161P5C5 protein of 8, 9, 10, or 11 amino acidresidues that bears an HLA Class I motif, or a subsequence of 9 or moreamino acid residues that bear an HLA Class II motif are within the scopeof the invention.

Expression of 161P5C5-Related Proteins

In an embodiment described in the examples that follow, 161P5C5 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 161P5C5 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 161P5C5 protein intransfected cells. The secreted HIS-tagged 161P5C5 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

Modifications of 161P5C5-Related Proteins

Modifications of 161P5C5-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a 161P5C5polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues of a161P5C5 protein. Another type of covalent modification of a 161P5C5polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.Another type of covalent modification of 161P5C5 comprises linking a161P5C5 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 161P5C5-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 161P5C5 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments of a161P5C5 sequence (amino or nucleic acid) such that a molecule is createdthat is not, through its length, directly homologous to the amino ornucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimericmolecule can comprise multiples of the same subsequence of 161P5C5. Achimeric molecule can comprise a fusion of a 161P5C5-related proteinwith a polyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind, with cytokines or with growthfactors. The epitope tag is generally placed at the amino- or carboxyl-terminus of a 161P5C5 protein. In an alternative embodiment, thechimeric molecule can comprise a fusion of a 161P5C5-related proteinwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a 161P5C5polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of anIgGI molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

Uses of 161P5C5-Related Proteins

The proteins of the invention have a number of different specific uses.As 161P5C5 is highly expressed in prostate and other cancers,161P5C5-related proteins are used in methods that assess the status of161P5C5 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of a 161P5C5 protein are used to assess the presence ofperturbations (such as deletions, insertions, point mutations etc.) inthose regions (such as regions containing one or more motifs). Exemplaryassays utilize antibodies or T cells targeting 161P5C5-related proteinscomprising the amino acid residues of one or more of the biologicalmotifs contained within a 161P5C5 polypeptide sequence in order toevaluate the characteristics of this region in normal versus canceroustissues or to elicit an immune response to the epitope. Alternatively,161P5C5-related proteins that contain the amino acid residues of one ormore of the biological motifs in a 161P5C5 protein are used to screenfor factors that interact with that region of 161P5C5.

161P5C5 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of a161P5C5 protein), for identifying agents or cellular factors that bindto 161P5C5 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 161P5C5 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to a 161P5C5 gene product.Antibodies raised against a 161P5C5 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 161P5C5protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 161P5C5-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

Various immunological assays useful for the detection of 161P5C5proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 161P5C5-expressingcells (e.g., in radioscintigraphic imaging methods). 161P5C5 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

161P5C5 Antibodies

Another aspect of the invention provides antibodies that bind to161P5C5-related proteins. Preferred antibodies specifically bind to a161P5C5-related protein and do not bind (or bind weakly) to peptides orproteins that are not 161P5C5-related proteins. For example, antibodiesthat bind 161P5C5 can bind 161P5C5-related proteins such as the homologsor analogs thereof.

161P5C5 antibodies of the invention are particularly useful in cancer(see, e.g., Table I) diagnostic and prognostic assays, and imagingmethodologies. Similarly, such antibodies are useful in the treatment,diagnosis, and/or prognosis of other cancers, to the extent 161P5C5 isalso expressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of161P5C5 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 161P5C5 and mutant 161P5C5-relatedproteins. Such assays can comprise one or more 161P5C5 antibodiescapable of recognizing and binding a 161P5C5-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 161P5C5 are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 161P5C5 antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 161P5C5 expressingcancers such as prostate cancer.

161P5C5 antibodies are also used in methods for purifying a161P5C5-related protein and for isolating 161P5C5 homologues and relatedmolecules. For example, a method of purifying a 161P5C5-related proteincomprises incubating a 161P5C5 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing a161P5C5-related protein under conditions that permit the 161P5C5antibody to bind to the 161P5C5-related protein; washing the solidmatrix to eliminate impurities; and eluting the 161P5C5-related proteinfrom the coupled antibody. Other uses of 161P5C5 antibodies inaccordance with the invention include generating anti-idiotypicantibodies that mimic a 161P5C5 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 161P5C5-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 161P5C5 canalso be used, such as a 161P5C5 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogento generate appropriate antibodies. In another embodiment, a161P5C5-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 161P5C5-related protein or 161P5C5 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly, et al., Ann. Rev. Immunol. (1997) 15: 617-648).

The amino acid sequence of a 161P5C5 protein as shown in FIG. 2 or FIG.3 can be analyzed to select specific regions of the 161P5C5 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of a 161P5C5 amino acid sequence are used to identifyhydrophilic regions in the 161P5C5 structure. Regions of a 161P5C5protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can begenerated using the method of Hopp, T. P. and Woods, K. R., Proc. Natl.Acad. Sci. U.S.A. (1981) 78:3824-3828. Hydropathicity profiles can begenerated using the method of Kyte, J. and Doolittle, R. F., J. Mol.Biol. (1982) 157:105-132. Percent (%) Accessible Residues profiles canbe generated using the method of Janin, J., Nature (1979) 277:491-492.Average Flexibility profiles can be generated using the method ofBhaskaran, R., and Ponnuswamy, P. K., Int. J. Pept. Protein Res. (1988)32:242-255. Beta-turn profiles can be generated using the method ofDeleage, G., and Roux B., Protein Engineering (1987) 1:289-294. Thus,each region identified by any of these programs or methods is within thescope of the present invention. Methods for the generation of 161P5C5antibodies are further illustrated by way of the examples providedherein. Methods for preparing a protein or polypeptide for use as animmunogen are well known in the art. Also well known in the art aremethods for preparing immunogenic conjugates of a protein with acarrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 161P5C5 immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

161P5C5 monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 161P5C5-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof a 161P5C5 protein can also be produced in the context of chimeric orcomplementarity determining region (CDR) grafted antibodies of multiplespecies origin. Humanized or human 161P5C5 antibodies can also beproduced, and are preferred for use in therapeutic contexts. Methods forhumanizing murine and other non-human antibodies, by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences, are well known (see for example, Jones, et al., Nature (1986)321: 522-525; Riechmann, et al., Nature (1988) 332: 323-327; Verhoeyen,et al., Science (1988) 239:1534-1536). See also, Carter, et al., Proc.Natl. Acad. Sci. USA (1993) 89:4285 and Sims, et al., J. Immunol. (1993)151:2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan, et al., NatureBiotechnology (1998) 16:535-539). Fully human 161P5C5 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies From Combinatorial Libraries. Id., pp. 65-82). Fully human161P5C5 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO 98/24893, Kucherlapati and Jakobovits, et al.,published Dec. 3, 1997 (see also, Jakobovits, Exp. Opin. Invest. Drugs(1998) 7:607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; 6,150,584issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598 issued 5 Sep. 2000).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of 161P5C5 antibodies with a 161P5C5-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,161P5C5-related proteins, 161P5C5-expressing cells or extracts thereof.A 161P5C5 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more161P5C5 epitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff, et al., Cancer Res. (1993) 53:2560-2565).

161P5C5 Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated.Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S., et al., Cell (1986)47:1071; Babbitt, B. P., et al., Nature (1985) 317:359; Townsend, A. andBodmer, H., Annu. Rev. Immunol. (1989) 7:601; Germain, R. N., Annu. Rev.Immunol. (1993) 11:403). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol. (1998) 160:3363; Rammensee, et al., Immunogenetics(1995) 41:178; Rammensee et al., SYFPEITHI, access via World Wide Web;Sette, A. and Sidney, J., Curr. Opin. Immunol. (1998) 10:478; Engelhard,V. H., Curr. Opin. Immunol. (1994) 6:13; Sette, A. and Grey, H. M.,Curr. Opin. Immunol. (1992) 4:79; Sinigaglia, F., and Hammer, J., Curr.Biol. (1994) 6:52; Ruppert, et al., Cell (1993) 74:929-937; Kondo, etal., J. Immunol. (1995) 155:4307-4312; Sidney, et al., J. Immunol.(1996) 157:3480-3490; Sidney, et al., Human Immunol. (1996) 45:79-93;Sette, A. and Sidney, J., Immunogenetics (1999) 50:201-212, Review).

Furthermore, X-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A. , Bjorkman, P. J. and Wiley, D. C., J. Mol.Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assayinvolving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol.8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). Forexample, in such methods peptides in incomplete Freund's adjuvant areadministered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a ⁵¹Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave been either effectively vaccinated and/or from chronically illpatients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995;Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin.Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648,1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly,recall responses are detected by culturing PBL from subjects that havebeen exposed to the antigen due to disease and thus have generated animmune response “naturally”, or from patients who were vaccinatedagainst the antigen. PBL from subjects are cultured in vitro for 1-2weeks in the presence of test peptide plus antigen presenting cells(APC) to allow activation of “memory” T cells, as compared to “naive” Tcells. At the end of the culture period, T cell activity is detectedusing assays including ⁵¹Cr release involving peptide-sensitizedtargets, T cell proliferation, or lymphokine release.

161P5C5 Transgenic Animals

Nucleic acids that encode a 161P5C5-related protein can also be used togenerate either transgenic animals or “knock out” animals that, in turn,are useful in the development and screening of therapeutically usefulreagents. In accordance with established techniques, cDNA encoding161P5C5 can be used to clone genomic DNA that encodes 161P5C5. Thecloned genomic sequences can then be used to generate transgenic animalscontaining cells that express DNA that encode 161P5C5. Methods forgenerating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat.No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would betargeted for 161P5C5 transgene incorporation with tissue-specificenhancers.

Transgenic animals that include a copy of a transgene encoding 161P5C5can be used to examine the effect of increased expression of DNA thatencodes 161P5C5. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this aspect ofthe invention, an animal is treated with a reagent and a reducedincidence of a pathological condition, compared to untreated animalsthat bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 161P5C5 can be used to constructa 161P5C5 “knock out” animal that has a defective or altered geneencoding 161P5C5 as a result of homologous recombination between theendogenous gene encoding 161P5C5 and altered genomic DNA encoding161P5C5 introduced into an embryonic cell of the animal. For example,cDNA that encodes 161P5C5 can be used to clone genomic DNA encoding161P5C5 in accordance with established techniques. A portion of thegenomic DNA encoding 161P5C5 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of a 161P5C5 polypeptide.

Methods for the Detection of 161P5C5

Another aspect of the present invention relates to methods for detecting161P5C5 polynucleotides and 161P5C5-related proteins, as well as methodsfor identifying a cell that expresses 161P5C5. The expression profile of161P5C5 makes it a diagnostic marker for metastasized disease.Accordingly, the status of 161P5C5 gene products provides informationuseful for predicting a variety of factors including susceptibility toadvanced stage disease, rate of progression, and/or tumoraggressiveness. As discussed in detail herein, the status of 161P5C5gene products in patient samples can be analyzed by a variety protocolsthat are well known in the art including immunohistochemical analysis,the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of161P5C5 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 161P5C5 polynucleotides include, for example, a 161P5C5gene or fragment thereof, 161P5C5 mRNA, alternative splice variant161P5C5 mRNAs, and recombinant DNA or RNA molecules that contain a161P5C5 polynucleotide. A number of methods for amplifying and/ordetecting the presence of 161P5C5 polynucleotides are well known in theart and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 161P5C5 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a161P5C5 polynucleotides as sense and antisense primers to amplify161P5C5 cDNAs therein; and detecting the presence of the amplified161P5C5 cDNA. Optionally, the sequence of the amplified 161P5C5 cDNA canbe determined.

In another embodiment, a method of detecting a 161P5C5 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 161P5C5 polynucleotides assense and antisense primers; and detecting the presence of the amplified161P5C5 gene. Any number of appropriate sense and antisense probecombinations can be designed from a 161P5C5 nucleotide sequence (see,e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of a161P5C5 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 161P5C5-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 161P5C5-related proteinin a biological sample comprises first contacting the sample with a161P5C5 antibody, a 161P5C5-reactive fragment thereof, or a recombinantprotein containing an antigen binding region of a 161P5C5 antibody; andthen detecting the binding of 161P5C5-related protein in the sample.

Methods for identifying a cell that expresses 161P5C5 are also withinthe scope of the invention. In one embodiment, an assay for identifyinga cell that expresses a 161P5C5 gene comprises detecting the presence of161P5C5 mRNA in the cell. Methods for the detection of particular mRNAsin cells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled 161P5C5 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for 161P5C5, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Alternatively, an assay for identifying a cell that expressesa 161P5C5 gene comprises detecting the presence of 161P5C5-relatedprotein in the cell or secreted by the cell. Various methods for thedetection of proteins are well known in the art and are employed for thedetection of 161P5C5-related proteins and cells that express161P5C5-related proteins.

161P5C5 expression analysis is also useful as a tool for identifying andevaluating agents that modulate 161P5C5 gene expression. For example,161P5C5 expression is significantly upregulated in prostate cancer, andis expressed in cancers of the tissues listed in Table I. Identificationof a molecule or biological agent that inhibits 161P5C5 expression orover-expression in cancer cells is of therapeutic value. For example,such an agent can be identified by using a screen that quantifies161P5C5 expression by RT-PCR, nucleic acid hybridization or antibodybinding.

Methods for Monitoring the Status of 161P5C5-Related Genes and TheirProducts

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant161P5C5 expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 161P5C5 in abiological sample of interest can be compared, for example, to thestatus of 161P5C5 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 161P5C5 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Greyer et al., J. Comp. Neurol.1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare161P5C5 status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 161P5C5 expressing cells) as well as the level, andbiological activity of expressed gene products (such as 161P5C5 mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of 161P5C5 comprises a change in the location of 161P5C5 and/or161P5C5 expressing cells and/or an increase in 161P5C5 mRNA and/orprotein expression.

161P5C5 status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of a 161P5C5 geneand gene products are found, for example in Ausubel et al. eds., 1995,Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4(Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus,the status of 161P5C5 in a biological sample is evaluated by variousmethods utilized by skilled artisans including, but not limited togenomic Southern analysis (to examine, for example perturbations in a161P5C5 gene), Northern analysis and/or PCR analysis of 161P5C5 mRNA (toexamine, for example alterations in the polynucleotide sequences orexpression levels of 161P5C5 mRNAs), and, Western and/orimmunohistochemical analysis (to examine, for example alterations inpolypeptide sequences, alterations in polypeptide localization within asample, alterations in expression levels of 161P5C5 proteins and/orassociations of 161P5C5 proteins with polypeptide binding partners).Detectable 161P5C5 polynucleotides include, for example, a 161P5C5 geneor fragment thereof, 161P5C5 mRNA, alternative splice variants, 161P5C5mRNAs, and recombinant DNA or RNA molecules containing a 161P5C5polynucleotide.

The expression profile of 161P5C5 makes it a diagnostic marker for localand/or metastasized disease, and provides information on the growth oroncogenic potential of a biological sample. In particular, the status of161P5C5 provides information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 161P5C5 status anddiagnosing cancers that express 161P5C5, such as cancers of the tissueslisted in Table I. For example, because 161P5C5 mRNA is so highlyexpressed in prostate and other cancers relative to normal prostatetissue, assays that evaluate the levels of 161P5C5 mRNA transcripts orproteins in a biological sample can be used to diagnose a diseaseassociated with 161P5C5 dysregulation, and can provide prognosticinformation useful in defining appropriate therapeutic options.

The expression status of 161P5C5 provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 161P5C5 inbiological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 161P5C5 in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of 161P5C5 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 161P5C5 expressing cells (e.g. those that express161P5C5 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth, for example, when 161P5C5-expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node), because such alterations in the status of161P5C5 in a biological sample are often associated with dysregulatedcellular growth. Specifically, one indicator of dysregulated cellulargrowth is the metastases of cancer cells from an organ of origin (suchas the prostate) to a different area of the body (such as a lymph node).In this context, evidence of dysregulated cellular growth is importantfor example because occult lymph node metastases can be detected in asubstantial proportion of patients with prostate cancer, and suchmetastases are associated with known predictors of disease progression(see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000); Su et al.,Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995Aug 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 161P5C5gene products by determining the status of 161P5C5 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 161P5C5gene products in a corresponding normal sample. The presence of aberrant161P5C5 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 161P5C5 mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 161P5C5 mRNA can, for example, beevaluated in tissues including but not limited to those listed in TableI. The presence of significant 161P5C5 expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express 161P5C5mRNA or express it at lower levels.

In a related embodiment, 161P5C5 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 161P5C5 protein expressed by cells ina test tissue sample and comparing the level so determined to the levelof 161P5C5 expressed in a corresponding normal sample. In oneembodiment, the presence of 161P5C5 protein is evaluated, for example,using immunohistochemical methods. 161P5C5 antibodies or bindingpartners capable of detecting 161P5C5 protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 161P5C5nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 161P5C5 may be indicative of the presence orpromotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 161P5C5 indicates a potential lossof function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 161P5C5 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S.Pat. No. 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of a 161P5C5 genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration ispresent in at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prey.,1998, 7:531-536). In another example, expression of the LAGE-I tumorspecific gene (which is not expressed in normal prostate but isexpressed in 25-50% of prostate cancers) is induced by deoxy-azacytidinein lymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). Avariety of assays for examining methylation status of a gene are wellknown in the art. For example, one can utilize, in Southernhybridization approaches, methylation-sensitive restriction enzymes thatcannot cleave sequences that contain methylated CpG sites to assess themethylation status of CpG islands. In addition, MSP (methylationspecific PCR) can rapidly profile the methylation status of all the CpGsites present in a CpG island of a given gene. This procedure involvesinitial modification of DNA by sodium bisulfite (which will convert allunmethylated cytosines to uracil) followed by amplification usingprimers specific for methylated versus unmethylated DNA. Protocolsinvolving methylation interference can also be found for example inCurrent Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel etal. eds., 1995.

Gene amplification is an additional method for assessing the status of161P5C5. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 161P5C5 expression. The presence of RT-PCRamplifiable 161P5C5 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting161P5C5 mRNA or 161P5C5 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 161P5C5 mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of 161P5C5 in prostate or other tissue isexamined, with the presence of 161P5C5 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 161P5C5 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 161P5C5 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 161P5C5 mRNA or 161P5C5 proteinexpressed by tumor cells, comparing the level so determined to the levelof 161P5C5 mRNA or 161P5C5 protein expressed in a corresponding normaltissue taken from the same individual or a normal tissue referencesample, wherein the degree of 161P5C5 mRNA or 161P5C5 protein expressionin the tumor sample relative to the normal sample indicates the degreeof aggressiveness. In a specific embodiment, aggressiveness of a tumoris evaluated by determining the extent to which 161P5C5 is expressed inthe tumor cells, with higher expression levels indicating moreaggressive tumors. Another embodiment is the evaluation of the integrityof 161P5C5 nucleotide and amino acid sequences in a biological sample,in order to identify perturbations in the structure of these moleculessuch as insertions, deletions, substitutions and the like. The presenceof one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 161P5C5 mRNA or161P5C5 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 161P5C5 mRNA or 161P5C5 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 161P5C5 mRNA or 161P5C5protein expression in the tumor sample over time provides information onthe progression of the cancer. In a specific embodiment, the progressionof a cancer is evaluated by determining 161P5C5 expression in the tumorcells over time, where increased expression over time indicates aprogression of the cancer. Also, one can evaluate the integrity 161P5C5nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 161P5C5 gene and161P5C5 gene products (or perturbations in 161P5C5 gene and 161P5C5 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSA, PSCA andPSM expression for prostate cancer etc.) as well as gross cytologicalobservations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al.,1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of 161P5C5 gene and 161P5C5 gene products (or perturbationsin 161P5C5 gene and 161P5C5 gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 161P5C5 gene and 161P5C5 gene products (or perturbationsin 161P5C5 gene and 161P5C5 gene products) and another factor associatedwith malignancy entails detecting the overexpression of 161P5C5 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample (or PSCA or PSM expression), and observing acoincidence of 161P5C5 mRNA or protein and PSA mRNA or proteinoverexpression (or PSCA or PSM expression). In a specific embodiment,the expression of 161P5C5 and PSA mRNA in prostate tissue is examined,where the coincidence of 161P5C5 and PSA mRNA overexpression in thesample indicates the existence of prostate cancer, prostate cancersusceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 161P5C5 mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 161P5C5 mRNAinclude in situ hybridization using labeled 161P5C5 riboprobes, Northernblot and related techniques using 161P5C5 polynucleotide probes, RT-PCRanalysis using primers specific for 161P5C5, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR is usedto detect and quantify 161P5C5 mRNA expression. Any number of primerscapable of amplifying 161P5C5 can be used for this purpose, includingbut not limited to the various primer sets specifically describedherein. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 161P5C5 protein can be used inan immunohistochemical assay of biopsied tissue.

Identification of Molecules That Interact With 161P5C5

The 161P5C5 protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with 161P5C5, as well as pathways activated by 161P5C5 viaany one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S.Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available inthe art for genome-based predictions of protein function (see, e.g.,Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 161P5C5 protein sequences. In such methods, peptidesthat bind to 161P5C5 are identified by screening libraries that encode arandom or controlled collection of amino acids. Peptides encoded by thelibraries are expressed as fusion proteins of bacteriophage coatproteins, the bacteriophage particles are then screened against the161P5C5 protein(s).

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 161P5C5 proteinsequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issued 3Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 161P5C5 are used to identifyprotein-protein interactions mediated by 161P5C5. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton B.J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 161P5C5protein can be immunoprecipitated from 161P5C5-expressing cell linesusing anti-161P5C5 antibodies. Alternatively, antibodies against His-tagcan be used in a cell line engineered to express fusions of 161P5C5 anda His-tag (vectors mentioned above). The immunoprecipitated complex canbe examined for protein association by procedures such as Westernblotting, ³⁵S-methionine labeling of proteins, protein microsequencing,silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 161P5C5 can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 161P5C5's ability to mediatephosphorylation and de-phosphorylation, interaction with DNA or RNAmolecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 161P5C5-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses 161P5C5 (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate 161P5C5 function can beidentified based on their ability to bind 161P5C5 and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of161P5C5 and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators which activate or inhibit 161P5C5.

An embodiment of this invention comprises a method of screening for amolecule that interacts with a 161P5C5 amino acid sequence shown in FIG.2 or FIG. 3, comprising the steps of contacting a population ofmolecules with a 161P5C5 amino acid sequence, allowing the population ofmolecules and the 161P5C5 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 161P5C5 amino acid sequence, and thenseparating molecules that do not interact with the 161P5C5 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 161P5C5 amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 161P5C5. In apreferred embodiment, the 161P5C5 amino acid sequence is contacted witha library of peptides.

Therapeutic Methods and Compositions

The identification of 161P5C5 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in prostate andother cancers, opens a number of therapeutic approaches to the treatmentof such cancers. As contemplated herein, 161P5C5 functions as atranscription factor involved in activating tumor-promoting genes orrepressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of a161P5C5 protein are useful for patients suffering from a cancer thatexpresses 161P5C5. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of a 161P5C5 protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of a 161P5C5 gene or translation of 161P5C5mRNA.

Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 161P5C5-relatedprotein or 161P5C5-related nucleic acid. In view of the expression of161P5C5, cancer vaccines prevent and/or treat 161P5C5-expressing cancerswith minimal or no effects on non-target tissues. The use of a tumorantigen in a vaccine that generates humoral and/or cell-mediated immuneresponses as anti-cancer therapy is well known in the art and has beenemployed in prostate cancer using human PSMA and rodent PAP immunogens(Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J.Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 161P5C5-relatedprotein, or a 161P5C5-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 161P5C5 immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 February 31(1):66-78; Maruyama et al.,Cancer Immunol Immunother 2000 June 49(3):123-32) Briefly, such methodsof generating an immune response (e.g. humoral and/or cell-mediated) ina mammal, comprise the steps of: exposing the mammal's immune system toan immunoreactive epitope (e.g. an epitope present in a 161P5C5 proteinshown in FIG. 3 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, a 161P5C5 immunogen contains a biological motif, seee.g., Tables V-XVIII and XXII-LI, or a peptide of a size range from161P5C5 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 161P5C5 protein, immunogenic regions or epitopes thereof canbe combined and delivered by various means. Such vaccine compositionscan include, for example, lipopeptides (e.g., Vitiello, A. et al., J.Clin. Invest. 95:341, 1995), peptide compositions encapsulated inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge,et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin ExpImmunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass. ) may also be used.

In patients with 161P5C5-associated cancer, the vaccine compositions ofthe invention can also be used in conjunction with other treatments usedfor cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identifypeptides within 161P5C5 protein that bind corresponding HLA alleles (seee.g., Table IV; Epimer™ and Epimatrix™, Brown University; and, BIMAS. Ina preferred embodiment, a 161P5C5 immunogen contains one or more aminoacid sequences identified using techniques well known in the art, suchas the sequences shown in Tables V-XVIII and XXII-LI or a peptide of 8,9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif(e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide ofat least 9 amino acids that comprises an HLA Class II motif/supermotif(e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, theHLA Class I binding groove is essentially closed ended so that peptidesof only a particular size range can fit into the groove and be bound,generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. Incontrast, the HLA Class II binding groove is essentially open ended;therefore a peptide of about 9 or more amino acids can be bound by anHLA Class II molecule. Due to the binding groove differences between HLAClass I and II, HLA Class I motifs are length specific, i.e., positiontwo of a Class I motif is the second amino acid in an amino to carboxyldirection of the peptide. The amino acid positions in a Class II motifare relative only to each other, not the overall peptide, i.e.,additional amino acids can be attached to the amino and/or carboxyltermini of a motif-bearing sequence. HLA Class II epitopes are often 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aminoacids long, or longer than 25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. a 161P5C5 protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 161P5C5 in a host, by contacting the host with asufficient amount of at least one 161P5C5 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 161P5C5 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 161P5C5-related protein or aman-made multiepitopic peptide comprising: administering 161P5C5immunogen (e.g. a 161P5C5 protein or a peptide fragment thereof, a161P5C5 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 161P5C5 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes a 161P5C5 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered. In addition, an antiidiotypic antibody can beadministered that mimics 161P5C5, in order to generate a response to thetarget antigen.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA that encode protein(s) of the invention can beadministered to a patient. Genetic immunization methods can be employedto generate prophylactic or therapeutic humoral and cellular immuneresponses directed against cancer cells expressing 161P5C5. Constructscomprising DNA encoding a 161P5C5-related protein/immunogen andappropriate regulatory sequences can be injected directly into muscle orskin of an individual, such that the cells of the muscle or skin take-upthe construct and express the encoded 161P5C5 protein/immunogen.Alternatively, a vaccine comprises a 161P5C5-related protein. Expressionof the 161P5C5-related protein immunogen results in the generation ofprophylactic or therapeutic humoral and cellular immunity against cellsthat bear a 161P5C5 protein. Various prophylactic and therapeuticgenetic immunization techniques known in the art can be used. Nucleicacid-based delivery is described, for instance, in Wolff et. al.,Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466;5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples ofDNA-based delivery technologies include “naked DNA”, facilitated(bupivicaine, polymers, peptide-mediated) delivery, cationic lipidcomplexes, and particle-mediated (“gene gun”) or pressure-mediateddelivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol.8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)).Non-viral delivery systems can also be employed by introducing naked DNAencoding a 161P5C5-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a 161P5C5-relatednucleic acid molecule. In one embodiment, the full-length human 161P5C5cDNA is employed. In another embodiment, 161P5C5 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells (DC) to present 161P5C5 antigen to apatient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent 161P5C5 peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed with161P5C5 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 161P5C5 protein. Yet another embodiment involves engineeringthe overexpression of a 161P5C5 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 161P5C5 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

161P5C5 as a Target for Antibody-Based Therapy

161P5C5 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 161P5C5 is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of161P5C5-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 161P5C5 areuseful to treat 161P5C5-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

161P5C5 antibodies can be introduced into a patient such that theantibody binds to 161P5C5 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 161P5C5,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of a 161P5C5 sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 161P5C5), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-161P5C5 antibody) that binds to a marker (e.g. 161P5C5)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing 161P5C5, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to a161P5C5 epitope, and, exposing the cell to the antibody-agent conjugate.Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-161P5C5 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin or radioisotope, such as the conjugation ofY⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDECPharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Theantibodies can be conjugated to a therapeutic agent. To treat prostatecancer, for example, 161P5C5 antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation. Also,antibodies can be conjugated to a toxin such as calicheamicin (e.g.,Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG₄kappa antibody conjugated to antitumor antibiotic calicheamicin) or amaytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform,ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

Although 161P5C5 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

Although 161P5C5 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 161P5C5expression, preferably using immunohistochemical assessments of tumortissue, quantitative 161P5C5 imaging, or other techniques that reliablyindicate the presence and degree of 161P5C5 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-161P5C5 monoclonal antibodies that treat prostate and other cancersinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, anti-161P5C5monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-161P5C5 mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 161P5C5. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-161P5C5 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 161P5C5antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-161P5C5 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-161P5C5 mAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-161P5C5 mAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-161P5C5 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-161P5C5antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-161P5C5 mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose is administeredas a 30 minute or longer infusion, provided the initial dose was welltolerated. As appreciated by those of skill in the art, various factorscan influence the ideal dose regimen in a particular case. Such factorsinclude, for example, the binding affinity and half life of the Ab ormAbs used, the degree of 161P5C5 expression in the patient, the extentof circulating shed 161P5C5 antigen, the desired steady-state antibodyconcentration level, frequency of treatment, and the influence ofchemotherapeutic or other agents used in combination with the treatmentmethod of the invention, as well as the health status of a particularpatient.

Optionally, patients should be evaluated for the levels of 161P5C5 in agiven sample (e.g. the levels of circulating 161P5C5 antigen and/or161P5C5 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-161P5C5 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 161P5C5-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-161P5C5 antibodiesthat mimic an epitope on a 161P5C5-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

161P5C5 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly L-lysine,poly L-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis, J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 161P5C5 antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I this includes 3-4 epitopes that come from at least onetumor associated antigen (TAA). For HLA Class II a similar rationale isemployed; again 3-4 epitopes are selected from at least one TAA (see,e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAAmay be used in combination with epitopes from one or more additionalTAAs to produce a vaccine that targets tumors with varying expressionpatterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene,an objective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

7.) Where the sequences of multiple variants of the same target proteinare present, potential peptide epitopes can also be selected on thebasis of their conservancy. For example, a criterion for conservancy maydefine that the entire sequence of an HLA class I binding peptide or theentire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka etal., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived 161P5C5, the PADRE®universal helper T cell epitope or multiple HTL epitopes from 161P5C5,(see e.g., Tables V-XVIII and XXII to LI), and an endoplasmicreticulum-translocating signal sequence can be engineered. A vaccine mayalso comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Felgner, et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ IDNO:24), Plasmodium falciparum circumsporozoite (CS) protein at positions378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO:25), and Streptococcus 18 kDprotein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO:26). Otherexamples include peptides bearing a DR 1-4-7 supermotif, or either ofthe DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed to most preferably bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: aKXVAAWTLKAAa (SEQ ID NO:27), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanineor L-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type. An alternative of a pan-DR binding epitopecomprises all “L” natural amino acids and can be provided in the form ofnucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include D-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini.

Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε- and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to specifically primean immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP₃CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to 161P5C5. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses 161P5C5.

Adoptive Immunotherapy

Antigenic 161P5C5-related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpresses161P5C5. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 161P5C5. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of 161P5C5-associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells)delivered to the patient may vary according to the stage of the diseaseor the patient's health status. For example, in a patient with a tumorthat expresses 161P5C5, a vaccine comprising 161P5C5-specific CTL may bemore efficacious in killing tumor cells in patient with advanced diseasethan alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to effectivelystimulate a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patient'sresponse and condition as determined by measuring the specific activityof CTL and HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g. as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-10⁷to 5×10⁹ pfu.

For antibodies, a treatment generally involves repeated administrationof the anti-161P5C5 antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. In general,doses in the range of 10-500 mg mAb per week are effective and welltolerated. Moreover, an initial loading dose of approximately 4 mg/kgpatient body weight IV, followed by weekly doses of about 2 mg/kg IV ofthe anti-161P5C5 mAb preparation represents an acceptable dosingregimen. As appreciated by those of skill in the art, various factorscan influence the ideal dose in a particular case. Such factors include,for example, half life of a composition, the binding affinity of an Ab,the immunogenicity of a substance, the degree of 161P5C5 expression inthe patient, the extent of circulating shed 161P5C5 antigen, the desiredsteady-state concentration level, frequency of treatment, and theinfluence of chemotherapeutic or other agents used in combination withthe treatment method of the invention, as well as the health status of aparticular patient. Non-limiting preferred human unit doses are, forexample, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg,800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, thedose is in a range of 2-5 mg/kg body weight, e.g., with follow on weeklydoses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg bodyweight followed, e.g., in two, three or four weeks by weekly doses;0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks byweekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m² of body areaweekly; 1-600 mg m² of body area weekly; 225-400 mg m² of body areaweekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7,8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art a therapeuticeffect depends on a number of factors, including the sequence of thepolynucleotide, molecular weight of the polynucleotide and route ofadministration. Dosages are generally selected by the physician or otherhealth care professional in accordance with a variety of parametersknown in the art, such as severity of symptoms, history of the patientand the like. Generally, for a polynucleotide of about 20 bases, adosage range may be selected from, for example, an independentlyselected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to anindependently selected upper limit, greater than the lower limit, ofabout 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dosemay be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg,0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg.Generally, parenteral routes of administration may require higher dosesof polynucleotide compared to more direct application to the nucleotideto diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitabledosage range or effective amount that provides any therapeutic effect.As appreciated by one of ordinary skill in the art, a therapeutic effectdepends on a number of factors. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. A dose may be about 10⁴ cells to about 10⁶cells, about 10⁶ cells to about 10⁸ cells, about 10⁸ to about 10¹¹cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸cells/m².

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01%-20% by weight, preferably about1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

Diagnostic and Prognostic Embodiments of 161P5C5.

As disclosed herein, 161P5C5 polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in the Example entitled “Expression analysis of 161P5C5 innormal tissues, and patient specimens”).

161P5C5 can be analogized to a prostate associated antigen PSA, thearchetypal marker that has been used by medical practitioners for yearsto identify and monitor the presence of prostate cancer (see, e.g.,Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J.Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. CancerInst. 91(19): 1635-1640(1999)). A variety of other diagnostic markersare also used in similar contexts including p53 and K-ras (see, e.g.,Tulchinsky et al., Int J Mol Med 1999 Jul. 4(1):99-102 and Minimoto etal., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this disclosure of161P5C5 polynucleotides and polypeptides (as well as 161P5C5polynucleotide probes and anti-161P5C5 antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 161P5C5polynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays whichemploy, e.g., PSA polynucleotides, polypeptides, reactive T cells andantibodies. For example, just as PSA polynucleotides are used as probes(for example in Northern analysis, see, e.g., Sharief et al., Biochem.Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCRanalysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000))to observe the presence and/or the level of PSA mRNAs in methods ofmonitoring PSA overexpression or the metastasis of prostate cancers, the161P5C5 polynucleotides described herein can be utilized in the same wayto detect 161P5C5 overexpression or the metastasis of prostate and othercancers expressing this gene. Alternatively, just as PSA polypeptidesare used to generate antibodies specific for PSA which can then be usedto observe the presence and/or the level of PSA proteins in methods tomonitor PSA protein overexpression (see, e.g., Stephan et al., Urology55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g.,Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 161P5C5polypeptides described herein can be utilized to generate antibodies foruse in detecting 161P5C5 overexpression or the metastasis of prostatecells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 161P5C5polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 161P5C5-expressing cells (lymph node) is found tocontain 161P5C5-expressing cells such as the 161P5C5 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 161P5C5 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 161P5C5 or express 161P5C5 at adifferent level are found to express 161P5C5 or have an increasedexpression of 161P5C5 (see, e.g., the 161P5C5 expression in the cancerslisted in Table I and in patient samples etc. shown in the accompanyingFigures). In such assays, artisans may further wish to generatesupplementary evidence of metastasis by testing the biological samplefor the presence of a second tissue restricted marker (in addition to161P5C5) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res.Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,161P5C5 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in the Example entitled “Expression analysisof 161P5C5 in normal tissues, and patient specimens,” where a 161P5C5polynucleotide fragment is used as a probe to show the expression of161P5C5 RNAs in cancer cells. In addition, variant polynucleotidesequences are typically used as primers and probes for the correspondingmRNAs in PCR and Northern analyses (see, e.g., Sawai et al., FetalDiagn. Ther. 1996 Nov.-Dec. 11(6):407-13 and Current Protocols InMolecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds.,1995)). Polynucleotide fragments and variants are useful in this contextwhere they are capable of binding to a target polynucleotide sequence(e.g., a 161P5C5 polynucleotide shown in FIG. 2 or variant thereof)under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 161P5C5 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 161P5C5biological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. a 161P5C5 polypeptide shown in FIG. 3).

As shown herein, the 161P5C5 polynucleotides and polypeptides (as wellas the 161P5C5 polynucleotide probes and anti-161P5C5 antibodies or Tcells used to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers such as thoselisted in Table I. Diagnostic assays that measure the presence of161P5C5 gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as 161P5C5polynucleotides and polypeptides (as well as the 161P5C5 polynucleotideprobes and anti-161P5C5 antibodies used to identify the presence ofthese molecules) need to be employed to confirm a metastases ofprostatic origin.

Finally, in addition to their use in diagnostic assays, the 161P5C5polynucleotides disclosed herein have a number of other utilities suchas their use in the identification of oncogenetic associated chromosomalabnormalities in the chromosomal region to which the 161P5C5 gene maps(see the Example entitled “Chromosomal Mapping of 161P5C5” below).Moreover, in addition to their use in diagnostic assays, the161P5C5-related proteins and polynucleotides disclosed herein have otherutilities such as their use in the forensic analysis of tissues ofunknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28;80(1-2): 63-9).

Additionally, 161P5C5-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 161P5C5. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to a 161P5C5 antigen. Antibodies or othermolecules that react with 161P5C5 can be used to modulate the functionof this molecule, and thereby provide a therapeutic benefit.

Inhibition of 161P5C5 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 161P5C5 to its binding partner or its association withother protein(s) as well as methods for inhibiting 161P5C5 function.

Inhibition of 161P5C5 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 161P5C5 are introduced into 161P5C5expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-161P5C5 antibody is expressed intracellularly,binds to 161P5C5 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol.Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to precisely target the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 161P5C5 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 161P5C5 intrabodies in orderto achieve the desired targeting. Such 161P5C5 intrabodies are designedto bind specifically to a particular 161P5C5 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to a 161P5C5protein are used to prevent 161P5C5 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 161P5C5 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

Inhibition of 161P5C5 with Recombinant Proteins

In another approach, recombinant molecules bind to 161P5C5 and therebyinhibit 161P5C5 function. For example, these recombinant moleculesprevent or inhibit 161P5C5 from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 161P5C5specific antibody molecule. In a particular embodiment, the 161P5C5binding domain of a 161P5C5 binding partner is engineered into a dimericfusion protein, whereby the fusion protein comprises two 161P5C5 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgG1. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3domains and the hinge region, but not the C_(H)1 domain. Such dimericfusion proteins are administered in soluble form to patients sufferingfrom a cancer associated with the expression of 161P5C5, whereby thedimeric fusion protein specifically binds to 161P5C5 and blocks 161P5C5interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

Inhibition of 161P5C5 Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 161P5C5 gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 161P5C5 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 161P5C5gene comprises contacting the 161P5C5 gene with a 161P5C5 antisensepolynucleotide. In another approach, a method of inhibiting 161P5C5 mRNAtranslation comprises contacting a 161P5C5 mRNA with an antisensepolynucleotide. In another approach, a 161P5C5 specific ribozyme is usedto cleave a 161P5C5 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the 161P5C5 gene, such as 161P5C5 promoter and/orenhancer elements. Similarly, proteins capable of inhibiting a 161P5C5gene transcription factor are used to inhibit 161P5C5 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 161P5C5 by interferingwith 161P5C5 transcriptional activation are also useful to treat cancersexpressing 161P5C5. Similarly, factors that interfere with 161P5C5processing are useful to treat cancers that express 161P5C5. Cancertreatment methods utilizing such factors are also within the scope ofthe invention.

General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing 161P5C5(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 161P5C5 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 161P5C5 antisensepolynucleotides, ribozymes, factors capable of interfering with 161P5C5transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 161P5C5 to a bindingpartner, etc.

In vivo, the effect of a 161P5C5 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628 and U.S.Pat. No. 6,107,540 describe various xenograft models of human prostatecancer capable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

Kits

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a 161P5C5-related protein or a 161P5C5 geneor message, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acidmolecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

A label can be present on the container to indicate that the compositionis used for a specific therapy or non-therapeutic application, and canalso indicate directions for either in vivo or in vitro use, such asthose described above. Directions and or other information can also beincluded on an insert which is included with the kit.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 161P5C5 Gene

To isolate genes that are over-expressed in kidney cancer, theSuppression Subtractive Hybridization (SSH) procedure using cDNA derivedfrom kidney cancer patient tissues was used.

The 161P5C5 SSH cDNA sequence (see FIG. 1A) was derived from asubtraction consisting of a kidney cancer minus normal kidney and amixture of 9 normal tissues: stomach, skeletal muscle, lung, brain,liver, kidney, pancreas, small intestine and heart. By RT-PCR, the161P5C5 cDNA was identified as highly expressed in patient cancerspecimens, with no expression detected in normal tissues (FIG. 14).

In another experiment, to isolate genes expressed in bladder cancer, theSSH procedure using cDNA derived from bladder cancer patient tissues wasused. The 163P3C6 SSH cDNA sequence was derived from a subtractionconsisting of a bladder cancer minus normal bladder and a mixture of 9normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney,pancreas, small intestine and heart. The 163P3C6 SSH cDNA sequence of467 bp, listed in FIG. 1B, did not show homology to any known gene, butdid show homology to the 161P5C5 gene. It was concluded that 161P5C5 isthe same gene as 163P3C6, and that 161P5C5 was isolated from both kidneycancer and bladder cancer subtraction experiments.

Materials and Methods

Human Tissues:

The patient cancer and normal tissues were purchased from differentsources such as the NDRI (Philadelphia, Pa.). mRNA for some normaltissues were purchased from Clontech, Palo Alto, Calif.

RNA Isolation:

Tissues were homogenized in Trizol reagent (Life Technologies, GibcoBRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purifiedfrom total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Totaland mRNA were quantified by spectrophotometric analysis (O.D. 260/280nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA Synthesis Primer):

5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: 28)

Adaptor 1:

5′CTAATACGACTCACTATAGGGCTCGAGCGGCC (SEQ ID NO: 29) GCCCGGGCAG3′3′GGCCCGTCCTAG5′ (SEQ ID NO: 30)

Adaptor 2:

5′GTAATACGACTCACTATAGGGCAGCGTGGTCG (SEQ ID NO: 31) CGGCCGAG3′3′CGGCTCCTAG5′ (SEQ ID NO: 32)

PCR Primer 1:

5′CTAATACGACTCACTATAGGGC3′ (SEQ ID NO: 33)

Nested Primer (NP)1:

5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ ID NO: 34)

Nested Primer (NP)2:

5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 35)

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that are differentially expressed in bladdercancer. The SSH reaction utilized cDNA from bladder cancer and normaltissues.

The gene 161P5C5 was derived from a kidney cancer pool minus normaltissue cDNA subtraction and from a bladder cancer pool minus normaltissue cDNA subtraction. The SSH DNA sequences (FIG. 1) were identified.

The cDNA derived from of pool of normal tissues was used as the sourceof the “driver” cDNA, while the cDNA from a pool of bladder cancertissues was used as the source of the “tester” cDNA. Double strandedcDNAs corresponding to tester and driver cDNAs were synthesized from 2μg of poly(A)⁺ RNA isolated from the relevant xenograft tissue, asdescribed above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1ng of oligonucleotide DPNCDN as primer. First- and second-strandsynthesis were carried out as described in the Kit's user manualprotocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). Theresulting cDNA was digested with Dpn II for 3 hrs at 37° C. DigestedcDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digestedcDNA from the relevant tissue source (see above) with a mix of digestedcDNAs derived from the nine normal tissues: stomach, skeletal muscle,lung, brain, liver, kidney, pancreas, small intestine, and heart.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant tissue source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ IDNO:36) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO:37) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH 8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: Initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 b.p.β-actin bands from multiple tissues were compared by visual inspection.Dilution factors for the first strand cDNAs were calculated to result inequal β-actin band intensities in all tissues after 22 cycles of PCR.Three rounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 161P5C5 gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification. Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities. The primers used for RT-PCR were designed using the 161P5C5SSH sequence and are listed below:

161P5C5.1 5′-GATATGCAGGAGGACACATTCTTG-3′ (SEQ ID NO: 38) 161P5C5.25′-GCTTCAGTAGGCAATGGTTTGATG-3′ (SEQ ID NO: 39) 163P3C6.15′-AGCTTGCTTCAATAGGCAATGGTT-3′ (SEQ ID NO: 40) 163P3C6.25′-TGCTATCTAAGTTGGAGGTTCTGCT-3′ (SEQ ID NO: 41)

A typical RT-PCR expression analysis is shown in FIG. 14. First strandcDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool2 (pancreas, colon and stomach), bladder cancer pool, kidney cancerpool, lung cancer pool, ovary cancer pool, breast cancer pool, andcancer metastasis pool. Normalization was performed by PCR using primersto actin and GAPDH. Semi-quantitative PCR, using primers to 161P5C5, wasperformed at 26 and 30 cycles of amplification. Results show strongexpression of 161P5C5 in bladder cancer pool, kidney cancer pool, ovarycancer pool, breast cancer pool, and cancer metastasis pool. Expressionof 161P5C5 was also detected in lung cancer pool, but not in vital pool1, and vital pool 2.

Example 2 The 161P5C5 SSH cDNA Sequence was Derived from a BladderCancer Pool Minus Normal Tissues cDNA Subtraction. The SSH cDNA Sequence(FIG. 1) was Designated 161P5C5

The SSH DNA sequence of 95 by (FIG. 1A) did not show homology to anyknown gene. The full-length cDNA 161P5C5 was cloned from bladder cancercDNA. Variants of 161P5C5 were identified and these are listed in FIGS.2 and 3. 161P5C5 gene is novel and did not show homology to any knowngenes.

Example 3 Chromosomal Mapping of 161P5C5

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available includingfluorescent in situ hybridization (FISH), human/hamster radiation hybrid(RH) panels (Walter et al., 1994; Nature Genetics 7:22; ResearchGenetics, Huntsville Ala.), human-rodent somatic cell hybrid panels suchas is available from the Coriell Institute (Camden, N.J.), and genomicviewers utilizing BLAST homologies to sequenced and mapped genomicclones (NCBI, Bethesda, Md.).

161P5C5 Maps to chromosome 7p21.1 using 161P5C5 sequence and the NCBIBLAST tool.

Example 4 Expression Analysis of 161P5C5 in Normal Tissues and PatientSpecimens

Expression analysis by RT-PCR demonstrated that 161P5C5 is stronglyexpressed in cancer patient specimens (FIG. 14). First strand cDNA wasprepared from vital pool 1 (liver, lung and kidney), vital pool 2(pancreas, colon and stomach), bladder cancer pool, kidney cancer pool,lung cancer pool, ovary cancer pool, breast cancer pool, and cancermetastasis pool. Normalization was performed by PCR using primers toactin and GAPDH. Semi-quantitative PCR, using primers to 161P5C5, wasperformed at 26 and 30 cycles of amplification. Results show strongexpression of 161P5C5 in bladder cancer pool, kidney cancer pool, ovarycancer pool, breast cancer pool, and cancer metastasis pool. Expressionof 161P5C5 was also detected in lung cancer pool, but not in vital pool1, and vital pool 2.

Extensive northern blot analysis of 161P5C5 in multiple human normaltissues is shown in FIG. 15. Two multiple tissue northern blots(Clontech) both with 2 μg of mRNA/lane were probed with the 161P5C5sequence. Size standards in kilobases (kb) are indicated on the side.Results show absence of expression of 161P5C5 in all 16 normal tissuestested.

Expression of 161P5C5 in patient bladder cancer specimens is shown inFIG. 16. RNA was extracted from normal bladder (NB), bladder cancer celllines (UM-UC-3, J82 and SCaBER), bladder cancer patient tumors (T) andnormal tissue adjacent to bladder cancer (N). Northern blots with 10 μgof total RNA were probed with the 161P5C5 SSH fragment. Size standardsin kilobases are indicated on the side. Results show strong expressionof 161P5C5 in the bladder tumor tissues but not in normal bladder, norin the bladder cancer cell lines.

FIG. 17 shows that 161P5C5 was expressed in kidney cancer patientspecimens. RNA was extracted from normal kidney (NK), kidney cancerpatient tumors (T) and their normal adjacent tissues (N). Northern blotswith 10 μg of total RNA were probed with the 161P5C5 SSH sequence.Results show strong expression of 161P5C5 in patient kidney cancertissues, but not in normal kidney.

Expression of 161P5C5 was also detected in ovary cancer patientspecimens (FIG. 18). RNA was extracted from ovary and cervical cancercell lines (CL), normal ovary (N), and ovary cancer patient tumors (T).Northern blots with 10 μg of total RNA were probed with the 161P5C5 SSHsequence. Results show strong expression of 161P5C5 in patient ovarycancer tissues, but not in normal ovary nor in the ovary and cervicalcancer cell lines.

The restricted expression of 161P5C5 in normal tissues and theexpression detected in bladder cancer, breast cancer, and cancermetastases indicate that 161P5C5 is a potential therapeutic target and adiagnostic marker for human cancers.

Example 5 Transcript Variants of 161P5C5

Transcript variants are variants of mature mRNA from the same gene whicharise by alternative transcription or alternative splicing. Alternativetranscripts are transcripts from the same gene but start transcriptionat different points. Splice variants are mRNA variants spliceddifferently from the same transcript. In eukaryotes, when a multi-exongene is transcribed from genomic DNA, the initial RNA is spliced toproduce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternative transcripts and each transcript can havezero to many splice variants. Each transcript variant has a unique exonmakeup, and can have different coding and/or non-coding (5′ or 3′ end)portions, from the original transcript. Transcript variants can code forsimilar or different proteins with the same or a similar function or canencode proteins with different functions, and can be expressed in thesame tissue at the same time, or in different tissues at the same time,or in the same tissue at different times, or in different tissues atdifferent times. Proteins encoded by transcript variants can havesimilar or different cellular or extracellular localizations, e.g.,secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods.For example, alternative transcripts and splice variants are identifiedby full-length cloning experiments, or by use of full-length transcriptand EST sequences. First, all human ESTs were grouped into clusterswhich show direct or indirect identity with each other. Second, ESTs inthe same cluster were further grouped into sub-clusters and assembledinto a consensus sequence. The original gene sequence is compared to theconsensus sequence(s) or other full-length sequences. Each consensussequence is a potential splice variant for that gene. Even when avariant is identified that is not a full-length clone, that portion ofthe variant is very useful for antigen generation and for furthercloning of the full-length splice variant, using techniques known in theart.

Moreover, computer programs are available in the art that identifytranscript variants based on genomic sequences. Genomic-based transcriptvariant identification programs include FgenesH (A. Salamov and V.Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” GenomeResearch. 2000 April; 10(4):516-22); Grail and GenScan. For a generaldiscussion of splice variant identification protocols see., e.g.,Southan, C., A genomic perspective on human proteases, FEBS Lett. 2001Jun. 8; 498(2-3):214-8; de Souza, S. J., et al., Identification of humanchromosome 22 transcribed sequences with ORF expressed sequence tags,Proc. Natl Acad Sci USA. 2000 Nov. 7; 97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety oftechniques are available in the art, such as full-length cloning,proteomic validation, PCR-based validation, and 5′ RACE validation, etc.(see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin bankspeninsula: a new termination variant characterized by electrospray massspectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6;Ferranti P, et al., Differential splicing of pre-messenger RNA producesmultiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. 1997Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al.,Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCyclertechnology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al.,Discovery of new human beta-defensins using a genomics-based approach,Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACEValidation: Brigle, K. E., et al., Organization of the murine reducedfolate carrier gene and identification of variant splice forms, BiochemBiophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers.When the genomic region to which a gene maps is modulated in aparticular cancer, the alternative transcripts or splice variants of thegene are modulated as well. Disclosed herein is that 161P5C5 has aparticular expression profile related to cancer. Alternative transcriptsand splice variants of 161P5C5 may also be involved in cancers in thesame or different tissues, thus serving as tumor-associatedmarkers/antigens.

The exon composition of the original transcript, designated as 161P5C5v.1, is shown in Table LIII. Using the full-length gene and ESTsequences, one splice variant was identified, designated as 161P5C5 v.7.Compared with 161P5C5 v.1, splice variant 161P5C5 v.7 spliced out exon2. In fact, each different combination of exons in spatial order, e.g.exons 2 and 3, is a potential splice variant. FIG. 12 shows theschematic alignment of exons of the two transcripts.

Table LIV shows nucleotide sequence of a transcript variant. Table LVshows the alignment of the transcript variant with nucleic acid sequenceof 161P5C5 v.1. Table LVI lays out amino acid translation of thetranscript variant for the identified reading frame orientation. TableLVII displays alignments of the amino acid sequence encoded by thesplice variant with that of 161P5C5 v.1.

Example 6 Single Nucleotide Polymorphisms of 161P5C5

A Single Nucleotide Polymorphism (SNP) is a single base pair variationin a nucleotide sequence at a specific location. At any given point ofthe genome, there are four possible nucleotide base pairs: A/T, C/G, G/Cand T/A. Genotype refers to the specific base pair sequence of one ormore locations in the genome of an individual. Haplotype refers to thebase pair sequence of more than one location on the same DNA molecule(or the same chromosome in higher organisms), often in the context ofone gene or in the context of several tightly linked genes. SNPs thatoccur on a cDNA are called cSNPs. These cSNPs may change amino acids ofthe protein encoded by the gene and thus change the functions of theprotein. Some SNPs cause inherited diseases; others contribute toquantitative variations in phenotype and reactions to environmentalfactors including diet and drugs among individuals. Therefore, SNPsand/or combinations of alleles (called haplotypes) have manyapplications, including diagnosis of inherited diseases, determinationof drug reactions and dosage, identification of genes responsible fordiseases, and analysis of the genetic relationship between individuals(P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect humantraits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M.Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drugreactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H.Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotidepolymorphisms in the isolation of common disease genes,”Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephensand A. Windemuth, “The predictive power of haplotypes in clinicalresponse,” Pharmacogenomics. 2000 February; 1(1):15-26).

SNPs are identified by a variety of art-accepted methods (P. Bean, “Thepromising voyage of SNP target discovery,” Am. Clin. Lab. 2001October-November; 20(9):18-20; K. M. Weiss, “In search of humanvariation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enablinglarge-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies,” Clin. Chem. 2001 February;47(2):164-172). For example, SNPs are identified by sequencing DNAfragments that show polymorphism by gel-based methods such asrestriction fragment length polymorphism (RFLP) and denaturing gradientgel electrophoresis (DGGE). They can also be discovered by directsequencing of DNA samples pooled from different individuals or bycomparing sequences from different DNA samples. With the rapidaccumulation of sequence data in public and private databases, one candiscover SNPs by comparing sequences using computer programs (Z. Gu, L.Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting incyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified andgenotype or haplotype of an individual can be determined by a variety ofmethods including direct sequencing and high throughput microarrays (P.Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu.Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft,“High-throughput SNP genotyping with the Masscode system,” Mol. Diagn.2000 December; 5(4):329-340).

Using the methods described above, five SNPs were identified in theoriginal transcript, 161P5C5 v.1, at positions 967 (A/G), 999 (C/T),1192 (A/G), 1221 (G/C) and 1350 (G/T). The transcripts or proteins withalternative alleles were designated as variants 161P5C5 v.2, v.3, v.4,v.5, and v.6. FIG. 10 shows the schematic alignment of the nucleotidevariants. FIG. 11 shows the schematic alignment of protein variants,corresponding to nucleotide variants. Nucleotide variants that code forthe same amino acid sequence as variant 1 are not shown in FIG. 11.These alleles of the SNPs, though shown separately here, can occur indifferent combinations (haplotypes) and in any one of the transcriptvariants that contains the sequence context of the SNPs, e.g., 161P5C5v.7.

Example 7 Production of Recombinant 161p5c5 in Prokaryotic Systems

To express recombinant 161P5C5 and 161P5C5 variants in prokaryoticcells, the full or partial length 161P5C5 and 161P5C5 variant cDNAsequences are cloned into any one of a variety of expression vectorsknown in the art. One or more of the following regions of 161P5C5 areused: amino acids 1-71; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous aminoacids from 161P5C5, variants, or analogs thereof.

A. In vitro Transcription and Translation Constructs:

pCRII: To generate 161P5C5 sense and anti-sense RNA probes for RNA insitu investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of the 161P5C5 cDNA. ThepCRII vector has Sp6 and T7 promoters flanking the insert to drive thetranscription of 161P5C5 RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 161P5C5 at the RNA level. Transcribed 161P5C5 RNArepresenting the cDNA amino acid coding region of the 161P5C5 gene isused in in vitro translation systems such as the TnT™ CoupledReticulolysate System (Promega, Corp., Madison, Wisc.) to synthesize161P5C5 protein.

B. Bacterial Constructs:

pGEX Constructs: To generate recombinant 161P5C5 proteins in bacteriathat are fused to the Glutathione S-transferase (GST) protein, all orparts of the 161P5C5 cDNA protein coding sequence are cloned into thepGEX family of GST-fusion vectors (Amersham Pharmacia Biotech,Piscataway, N.J.). These constructs allow controlled expression ofrecombinant 161P5C5 protein sequences with GST fused at theamino-terminus and a six histidine epitope (6× His) at thecarboxyl-terminus. The GST and 6× His tags permit purification of therecombinant fusion protein from induced bacteria with the appropriateaffinity matrix and allow recognition of the fusion protein withanti-GST and anti-His antibodies. The 6× His tag is generated by adding6 histidine codons to the cloning primer at the 3′ end, e.g., of theopen reading frame (ORF). A proteolytic cleavage site, such as thePreScission™ recognition site in pGEX-6P-1, may be employed such that itpermits cleavage of the GST tag from 161P5C5-related protein. Theampicillin resistance gene and pBR322 origin permits selection andmaintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 161P5C5 proteinsthat are fused to maltose-binding protein (MBP), all or parts of the161P5C5 cDNA protein coding sequence are fused to the MBP gene bycloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs,Beverly, Mass.). These constructs allow controlled expression ofrecombinant 161P5C5 protein sequences with MBP fused at theamino-terminus and a 6× His epitope tag at the carboxyl-terminus. TheMBP and 6× His tags permit purification of the recombinant protein frominduced bacteria with the appropriate affinity matrix and allowrecognition of the fusion protein with anti-MBP and anti-His antibodies.The 6× His epitope tag is generated by adding 6 histidine codons to the3′ cloning primer. A Factor Xa recognition site permits cleavage of thepMAL tag from 161P5C5. The pMAL-c2X and pMAL-p2X vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds.

pET Constructs: To express 161P5C5 in bacterial cells, all or parts ofthe 161P5C5 cDNA protein coding sequence are cloned into the pET familyof vectors (Novagen, Madison, Wisc.). These vectors allow tightlycontrolled expression of recombinant 161P5C5 protein in bacteria withand without fusion to proteins that enhance solubility, such as NusA andthioredoxin (Trx), and epitope tags, such as 6× His and S-Tag™ that aidpurification and detection of the recombinant protein. For example,constructs are made utilizing pET NusA fusion system 43.1 such thatregions of the 161P5C5 protein are expressed as amino-terminal fusionsto NusA.

C. Yeast Constructs:

pESC Constructs: To express 161P5C5 in the yeast species Saccharomycescerevisiae for generation of recombinant protein and functional studies,all or parts of the 161P5C5 cDNA protein coding sequence are cloned intothe pESC family of vectors each of which contain 1 of 4 selectablemarkers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.).These vectors allow controlled expression from the same plasmid of up to2 different genes or cloned sequences containing either Flag™ or Mycepitope tags in the same yeast cell. This system is useful to confirmprotein-protein interactions of 161P5C5. In addition, expression inyeast yields similar post-translational modifications, such asglycosylations and phosphorylations, that are found when expressed ineukaryotic cells.

pESP Constructs: To express 161P5C5 in the yeast species Saccharomycespombe, all or parts of the 161P5C5 cDNA protein coding sequence arecloned into the pESP family of vectors. These vectors allow controlledhigh level of expression of a 161P5C5 protein sequence that is fused ateither the amino terminus or at the carboxyl terminus to GST which aidspurification of the recombinant protein. A Flag™ epitope tag allowsdetection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant 161P5C5 in Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 161P5C5 in eukaryotic cells, the full or partiallength 161P5C5 cDNA sequences can be cloned into any one of a variety ofexpression vectors known in the art. One or more of the followingregions of 161P5C5 are expressed in these constructs, amino acids 1 to71, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous amino acids from161P5C5, variants, or analogs thereof. In certain embodiments a regionof a specific variant of 161P5C5 is expressed that encodes an amino acidat a specific position which differs from the amino acid of any othervariant found at that position. In other embodiments, a region of avariant of 161P5C5 is expressed that lies partly or entirely within asequence that is unique to that variant.

The constructs can be transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-161P5C5 polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 161P5C5 in mammalian cells, a161P5C5 ORF, or portions thereof, of 161P5C5 are cloned intopcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter and theSP16 translational enhancer. The recombinant protein has Xpress™ and sixhistidine (6×His) epitopes fused to the amino-terminus. ThepcDNA4/HisMax vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs: To express 161P5C5 in mammalian cells, a161P5C5 ORF, or portions thereof, of 161P5C5 with a consensus Kozaktranslation initiation site are cloned into pcDNA3.1/MycHis Version A(Invitrogen, Carlsbad, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the mycepitope and 6×His epitope fused to the carboxyl-terminus. ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability, along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene can be used, as it allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli.

pcDNA3.1/CT-GFP-TOPO Construct: To express 161P5C5 in mammalian cellsand to allow detection of the recombinant proteins using fluorescence, a161P5C5 ORF, or portions thereof, with a consensus Kozak translationinitiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA).Protein expression is driven from the cytomegalovirus (CMV) promoter.The recombinant proteins have the Green Fluorescent Protein (GFP) fusedto the carboxyl-terminus facilitating non-invasive, in vivo detectionand cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also containsthe bovine growth hormone (BGH) polyadenylation signal and transcriptiontermination sequence to enhance mRNA stability along with the SV40origin for episomal replication and simple vector rescue in cell linesexpressing the large T antigen. The Neomycin resistance gene allows forselection of mammalian cells that express the protein, and theampicillin resistance gene and ColE1 origin permits selection andmaintenance of the plasmid in E. coli. Additional constructs with anamino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning theentire length of a 161P5C5 protein.

PAPtag: A 161P5C5 ORF, or portions thereof, is cloned into pAPtag-5(GenHunter Corp. Nashville, Tenn.). This construct generates an alkalinephosphatase fusion at the carboxyl-terminus of a 161P5C5 protein whilefusing the IgGκ signal sequence to the amino-terminus. Constructs arealso generated in which alkaline phosphatase with an amino-terminal IgGκsignal sequence is fused to the amino-terminus of a 161P5C5 protein. Theresulting recombinant 161P5C5 proteins are optimized for secretion intothe media of transfected mammalian cells and can be used to identifyproteins such as ligands or receptors that interact with 161P5C5proteins. Protein expression is driven from the CMV promoter and therecombinant proteins also contain myc and 6× His epitopes fused at thecarboxyl-terminus that facilitates detection and purification. TheZeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the recombinant protein and the ampicillinresistance gene permits selection of the plasmid in E. coli.

ptag5: A 161P5C5 ORF, or portions thereof, is cloned into pTag-5. Thisvector is similar to pAPtag but without the alkaline phosphatase fusion.This construct generates 161P5C5 protein with an amino-terminal IgGκsignal sequence and myc and 6× His epitope tags at the carboxyl-terminusthat facilitate detection and affinity purification. The resultingrecombinant 161P5C5 protein is optimized for secretion into the media oftransfected mammalian cells, and is used as immunogen or ligand toidentify proteins such as ligands or receptors that interact with the161P5C5 proteins. Protein expression is driven from the CMV promoter.The Zeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the protein, and the ampicillin resistancegene permits selection of the plasmid in E. coli.

PsecFc: A 161P5C5 ORF, or portions thereof, is also cloned into psecFc.The psecFc vector was assembled by cloning the human immunoglobulin G1(IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen,California). This construct generates an IgG1 Fc fusion at thecarboxyl-terminus of the 161P5C5 proteins, while fusing the IgGK signalsequence to N-terminus. 161P5C5 fusions utilizing the murine IgG1 Fcregion are also used. The resulting recombinant 161P5C5 proteins areoptimized for secretion into the media of transfected mammalian cells,and can be used as immunogens or to identify proteins such as ligands orreceptors that interact with 161P5C5 protein. Protein expression isdriven from the CMV promoter. The hygromycin resistance gene present inthe vector allows for selection of mammalian cells that express therecombinant protein, and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

pSRα Constructs: To generate mammalian cell lines that express 161P5C5constitutively, 161P5C5 ORF, or portions thereof, of 161P5C5 are clonedinto pSRα constructs. Amphotropic and ecotropic retroviruses aregenerated by transfection of pSRα constructs into the 293T-10A1packaging line or co-transfection of pSRα and a helper plasmid(containing deleted packaging sequences) into the 293 cells,respectively. The retrovirus is used to infect a variety of mammaliancell lines, resulting in the integration of the cloned gene, 161P5C5,into the host cell-lines. Protein expression is driven from a longterminal repeat (LTR). The Neomycin resistance gene present in thevector allows for selection of mammalian cells that express the protein,and the ampicillin resistance gene and ColE1 origin permit selection andmaintenance of the plasmid in E. coli. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as theFLAG™ tag to the carboxyl-terminus of 161P5C5 sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ (SEQ ID NO:42) is added to cloningprimer at the 3′ end of the ORF. Additional pSRα constructs are made toproduce both amino-terminal and carboxyl-terminal GFP and myc/6× Hisfusion proteins of the full-length 161P5C5 proteins.

Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 161P5C5. High virus titerleading to high level expression of 161P5C5 is achieved in viraldelivery systems such as adenoviral vectors and herpes amplicon vectors.A 161P5C5 coding sequences or fragments thereof are amplified by PCR andsubcloned into the AdEasy shuttle vector (Stratagene). Recombination andvirus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 161P5C5coding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as PC3, NIH3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 161P5C5 inmammalian cells, coding sequences of 161P5C5, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant161P5C5. These vectors are thereafter used to control expression of161P5C5 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 161P5C5 proteins in a baculovirus expressionsystem, 161P5C5 ORF, or portions thereof, are cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-161P5C5 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 161P5C5 protein is then generated by infection of HighFiveinsect cells (Invitrogen) with purified baculovirus. Recombinant 161P5C5protein can be detected using anti-161P5C5 or anti-His-tag antibody.161P5C5 protein can be purified and used in various cell-based assays oras immunogen to generate polyclonal and monoclonal antibodies specificfor 161P5C5.

Example 9 Antigenicity Profiles and Secondary Structure

FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict graphically five aminoacid profiles of the 161P5C5 variants 1 through 4 respectively, eachassessment available by accessing the ProtScale website located on theWorld Wide Web at (.expasy.ch/cgi-bin/protscale.pl) on the ExPasymolecular biology server.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981.Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity,(Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7,Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG.8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int.J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., RouxB. 1987 Protein Engineering 1:289-294); and optionally others availablein the art, such as on the ProtScale website, were used to identifyantigenic regions of the 161P5C5 protein. Each of the above amino acidprofiles of 161P5C5 were generated using the following ProtScaleparameters for analysis: 1) A window size of 9; 2) 100% weight of thewindow edges compared to the window center; and, 3) amino acid profilevalues normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profile, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determinestretches of amino acids (i.e., values greater than 0.5 on the Beta-turnprofile and the Average Flexibility profile) that are not constrained insecondary structures such as beta sheets and alpha helices. Such regionsare also more likely to be exposed on the protein and thus accessible toimmune recognition, such as by antibodies.

Antigenic sequences of the 161P5C5 protein indicated, e.g., by theprofiles set forth in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and/or FIG. 9 areused to prepare immunogens, either peptides or nucleic acids that encodethem, to generate therapeutic and diagnostic anti-161P5C5 antibodies.The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50contiguous amino acids, or the corresponding nucleic acids that encodethem, from the 161P5C5 protein variants listed in FIGS. 2 and 3. Inparticular, peptide immunogens of the invention can comprise: a peptideregion of at least 5 amino acids of FIGS. 2 and 3 in any whole numberincrement that includes an amino acid position having a value greaterthan 0.5 in the Hydrophilicity profiles of FIG. 5; a peptide region ofat least 5 amino acids of FIGS. 2 and 3 in any whole number incrementthat includes an amino acid position having a value less than 0.5 in theHydropathicity profile of FIG. 6; a peptide region of at least 5 aminoacids of FIGS. 2 and 3 in any whole number increment that includes anamino acid position having a value greater than 0.5 in the PercentAccessible Residues profiles of FIG. 7; a peptide region of at least 5amino acids of FIGS. 2 and 3 in any whole number increment that includesan amino acid position having a value greater than 0.5 in the AverageFlexibility profiles on FIG. 8; and, a peptide region of at least 5amino acids of FIGS. 2 and 3 in any whole number increment that includesan amino acid position having a value greater than 0.5 in the Beta-turnprofile of FIG. 9. Peptide immunogens of the invention can also comprisenucleic acids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 161P5C5 variant 1, namely the predictedpresence and location of alpha helices, extended strands, and randomcoils, is predicted from the primary amino acid sequence using theHNN—Hierarchical Neural Network method (Guermeur, 1997), accessed fromthe ExPasy molecular biology server. The analysis indicates that 161P5C5variant 1 is composed of 59.15% alpha helix, 12.68% extended strand, and28.17% random (FIG. 13).

Analysis for the potential presence of transmembrane domains in 161P5C5variant 1 was carried out using a variety of transmembrane predictionalgorithms accessed from the ExPasy molecular biology server located onthe World Wide Web. The results of the analysis are summarized in TableXXI, physical properties of 161P5C5. The majority of the programs do notpredict the presence of transmembrane domains in 161P5C5, suggestingthat it is a soluble protein.

Example 10 Generation of 161P5C5 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with the full length 161P5C5 protein, computeralgorithms are employed in design of immunogens that, based on aminoacid sequence analysis contain characteristics of being antigenic andavailable for recognition by the immune system of the immunized host(see the Example entitled “Antigenicity Profiles”). Such regions wouldbe predicted to be hydrophilic, flexible, in beta-turn conformations,and/or be exposed on the surface of the protein (see, e.g., FIG. 5, FIG.6, FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate suchregions of 161P5C5 and variants).

For example, 161P5C5 recombinant bacterial fusion proteins or peptidescontaining hydrophilic, flexible, beta-turn regions of 161P5C5 variantproteins are used as antigens to generate polyclonal antibodies in NewZealand White rabbits. For example, such regions include, but are notlimited to, amino acids 1-23 and 39-53 of variant 1. It is useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Examples of such immunogenic proteinsinclude, but are not limited to, keyhole limpet hemocyanin (KLH), serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor. In oneembodiment, a peptide encoding amino acids 1-23 of 161P5C5 variant 1 isconjugated to KLH and used to immunize the rabbit. Alternatively theimmunizing agent may include all or portions of the 161P5C5 variantproteins, analogs or fusion proteins thereof. For example, the 161P5C5variant 1 amino acid sequence can be fused using recombinant DNAtechniques to any one of a variety of fusion protein partners that arewell known in the art, such as glutathione-S-transferase (GST) and HIStagged fusion proteins. Such fusion proteins are purified from inducedbacteria using the appropriate affinity matrix.

In one embodiment, a GST-fusion protein encoding the full length 161P5C5variant 1 gene, amino acids 1-71, is produced and purified and used asimmunogen. Other recombinant bacterial fusion proteins that may beemployed include maltose binding protein, LacZ, thioredoxin, NusA, or animmunoglobulin constant region (see the section entitled “Production of161P5C5 in Prokaryotic Systems” and Current Protocols In MolecularBiology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995;Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., andLedbetter, L. (1991) J. Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the Tag5 and Fc-fusion vectors (seethe section entitled “Production of Recombinant 161P5C5 in EukaryoticSystems”), and retain post-translational modifications such asglycosylations found in native protein. In one embodiment, the fulllength sequence of variant 1, amino acids 1-71, is cloned into the Tag5mammalian secretion vector. The recombinant protein is purified by metalchelate chromatography from tissue culture supernatants of 293T cellsstably expressing the recombinant vector. The purified Tag5 161P5C5protein is then used as immunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbitserum derived from immunization with a KLH-conjugated peptide encodingamino acids 1-23 of variant 1, the full-length 161P5C5 variant 1 cDNA iscloned into pCDNA 3.1 myc-his expression vector (Invitrogen, see theExample entitled “Production of Recombinant 161P5C5 in EukaryoticSystems”). After transfection of the constructs into 293T cells, celllysates are probed with the anti-161P5C5 serum and with anti-Hisantibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determinespecific reactivity to denatured 161P5C5 protein using the Western blottechnique. In addition, the immune serum is tested by fluorescencemicroscopy, flow cytometry and immunoprecipitation against 293T andother recombinant 161P5C5-expressing cells to determine specificrecognition of native protein. Western blot, immunoprecipitation,fluorescent microscopy, and flow cytometric techniques using cells thatendogenously express 161P5C5 are also carried out to test reactivity andspecificity.

Anti-serum from rabbits immunized with 161P5C5 variant fusion proteins,such as GST and MBP fusion proteins, are purified by depletion ofantibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. For example, antiserum derivedfrom a GST-161P5C5 fusion protein encoding amino acids 1-71 is firstpurified by passage over a column of GST protein covalently coupled toAffiGel matrix (BioRad, Hercules, Calif.). The antiserum is thenaffinity purified by passage over a column composed of a MBP-fusionprotein also encoding amino acids 1-71 covalently coupled to Affigelmatrix. The serum is then further purified by protein G affinitychromatography to isolate the IgG fraction. Sera from other His-taggedantigens and peptide immunized rabbits as well as fusion partnerdepleted sera are affinity purified by passage over a column matrixcomposed of the original protein immunogen or free peptide.

Example 11 Generation of 161P5C5 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 161P5C5 variants comprise thosethat react with epitopes specific for each variant protein or specificto sequences in common between the variants that would disrupt ormodulate the biological function of the 161P5C5 variants, for examplethose that would disrupt the interaction with ligands and bindingpartners. Immunogens for generation of such mAbs include those designedto encode or contain the entire 161P5C5 protein variant sequence,regions of the 161P5C5 protein variants predicted to be antigenic fromcomputer analysis of the amino acid sequence (see, e.g., FIG. 5, FIG. 6,FIG. 7, FIG. 8, or FIG. 9, and the Example entitled “AntigenicityProfiles”). Immunogens include peptides, recombinant bacterial proteins,and mammalian expressed Tag 5 proteins and human and murine IgG FCfusion proteins. In addition, cells engineered to express high levels ofa respective 161P5C5 variant, such as 293T-161P5C5 variant 1 or300.19-161P5C5 variant 1 murine Pre-B cells, are used to immunize mice.

To generate mAbs to a 161P5C5 variant, mice are first immunizedintraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or10⁷ 161P5C5-expressing cells mixed in complete Freund's adjuvant. Miceare then subsequently immunized IP every 2-4 weeks with, typically,10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund'sadjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. Inaddition to the above protein and cell-based immunization strategies, aDNA-based immunization protocol is employed in which a mammalianexpression vector encoding a 161P5C5 variant sequence is used toimmunize mice by direct injection of the plasmid DNA. For example, thefull length variant 1 sequence, encoding amino acids 1-71, is clonedinto the Tag5 mammalian secretion vector and the recombinant vector isused as immunogen. In another example the same amino acids are clonedinto an Fc-fusion secretion vector in which the 161P5C5 variant 1sequence is fused at the amino-terminus to an IgK leader sequence and atthe carboxyl-terminus to the coding sequence of the human or murine IgGFc region. This recombinant vector is then used as immunogen. Theplasmid immunization protocols are used in combination with purifiedproteins expressed from the same vector and with cells expressing therespective 161P5C5 variant.

During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, and flow cytometric analyses, fusion and hybridomageneration is then carried out with established procedures well known inthe art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 161P5C5 monoclonal antibodies, aTag5-161P5C5 variant 1 antigen encoding amino acids 1-71, is expressedand purified from stably transfected 293T cells. Balb C mice areinitially immunized intraperitoneally with 25 μg of the Tag5-161P5C5variant 1 protein mixed in complete Freund's adjuvant. Mice aresubsequently immunized every two weeks with 25 μg of the antigen mixedin incomplete Freund's adjuvant for a total of three immunizations.ELISA using the Tag5 antigen determines the titer of serum fromimmunized mice. Reactivity and specificity of serum to full length161P5C5 variant 1 protein is monitored by Western blotting,immunoprecipitation and flow cytometry using 293T cells transfected withan expression vector encoding the 161P5C5 variant 1 cDNA (see e.g., theExample entitled “Production of Recombinant 161P5C5 in EukaryoticSystems”). Other recombinant 161P5C5 variant 1-expressing cells or cellsendogenously expressing 161P5C5 variant 1 are also used. Mice showingthe strongest reactivity are rested and given a final injection of Tag5antigen in PBS and then sacrificed four days later. The spleens of thesacrificed mice are harvested and fused to SPO/2 myeloma cells usingstandard procedures (Harlow and Lane, 1988). Supernatants from HATselected growth wells are screened by ELISA, Western blot,immunoprecipitation, fluorescent microscopy, and flow cytometry toidentify 161P5C5 specific antibody-producing clones.

The binding affinity of a 161P5C5 monoclonal antibody is determinedusing standard technologies. Affinity measurements quantify the strengthof antibody to epitope binding and are used to help define which 161P5C5monoclonal antibodies preferred for diagnostic or therapeutic use, asappreciated by one of skill in the art. The BIAcore system (Uppsala,Sweden) is a preferred method for determining binding affinity. TheBIAcore system uses surface plasmon resonance (SPR, Welford K. 1991,Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology295: 268) to monitor biomolecular interactions in real time. BIAcoreanalysis conveniently generates association rate constants, dissociationrate constants, equilibrium dissociation constants, and affinityconstants.

Example 12 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995);Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], the measuredIC₅₀ values are reasonable approximations of the true K_(D) values.Peptide inhibitors are typically tested at concentrations ranging from120 μg/ml to 1.2 ng/ml, and are tested in two to four completelyindependent experiments. To allow comparison of the data obtained indifferent experiments, a relative binding figure is calculated for eachpeptide by dividing the IC₅₀ of a positive control for inhibition by theIC₅₀ for each tested peptide (typically unlabeled versions of theradiolabeled probe peptide). For database purposes, and inter-experimentcomparisons, relative binding values are compiled. These values cansubsequently be converted back into IC₅₀ nM values by dividing the IC₅₀nM of the positive controls for inhibition by the relative binding ofthe peptide of interest. This method of data compilation is accurate andconsistent for comparing peptides that have been tested on differentdays, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides (see Table IV).

Example 13 Identification of HLA Supermotif- and Motif-Bearing CTLCandidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes.The multiple epitopes can comprise multiple HLA supermotifs or motifs toachieve broad population coverage. This example illustrates theidentification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/orMotif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequencesin the Example entitled “Antigenicity Profiles” and Tables V-XVIII andXXII-LI employ the protein sequence data from the gene product of161P5C5 set forth in FIGS. 2 and 3; the specific peptides used togenerate the tables are listed in Table LII.

Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 161P5C5protein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or ΔG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:“ΔG”=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni)

where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (j) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount j_(i) to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has beendescribed in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (seealso Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al.,J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchorand non-anchor alike, the geometric mean of the average relative binding(ARB) of all peptides carrying j is calculated relative to the remainderof the group, and used as the estimate of j_(i). For Class II peptides,if multiple alignments are possible, only the highest scoring alignmentis utilized, following an iterative procedure. To calculate an algorithmscore of a given peptide in a test set, the ARB values corresponding tothe sequence of the peptide are multiplied. If this product exceeds achosen threshold, the peptide is predicted to bind. Appropriatethresholds are chosen as a function of the degree of stringency ofprediction desired.

Selection of HLA-A2 Supertype Cross-reactive Peptides

Protein sequences from 161P5C5 are scanned utilizing motifidentification software, to identify 8-, 9-10- and 11-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

These peptides are then tested for the capacity to bind to additionalA2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptidesthat bind to at least three of the five A2-supertype alleles tested aretypically deemed A2-supertype cross-reactive binders. Preferred peptidesbind at an affinity equal to or less than 500 nM to three or more HLA-A2supertype molecules.

Selection of HLA-A3 Supermotif-bearing Epitopes

The 161P5C5 protein sequence(s) scanned above is also examined for thepresence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of ≦500 nM, often ≦200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 161P5C5 protein(s) scanned above is also analyzed for the presenceof 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif.Corresponding peptides are synthesized and tested for binding toHLA-B*0702, the molecule encoded by the most common B7-supertype allele(i.e., the prototype B7 supertype allele). Peptides binding B*0702 withIC₅₀ of ≦500 nM are identified using standard methods. These peptidesare then tested for binding to other common B7-supertype molecules(e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of bindingto three or more of the five B7-supertype alleles tested are therebyidentified.

Selection of A1 and A24 Motif-bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes canalso be incorporated into vaccine compositions. An analysis of the161P5C5 protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear othermotif and/or supermotifs are identified using analogous methodology.

Example 14 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that areidentified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

Target Cell Lines for Cellular Screening:

The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene intothe HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221,is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640plus 5% AB human serum, non-essential amino acids, sodium pyruvate,L-glutamine and penicillin/streptomycin). The monocytes are purified byplating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at 37° C., thenon-adherent cells are removed by gently shaking the plates andaspirating the supernatants. The wells are washed a total of three timeswith 3 ml RPMI to remove most of the non-adherent and loosely adherentcells. Three ml of complete medium containing 50 ng/ml of GM-CSF and1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCson day 6 at 75 ng/ml and the cells are used for CTL induction cultureson day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated bypositive selection with Dynal immunomagnetic beads (Dynabeads® M-450)and the detacha-bead® reagent. Typically about 200-250×10⁶ PBMC areprocessed to obtain 24×10⁶ CD8⁺ T-cells (enough for a 48-well plateculture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×10⁶ cells/ml. The magneticbeads are washed 3 times with PBS/AB serum, added to the cells (140 μlbeads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4× with PBS/AB serum to removethe nonadherent cells and resuspended at 100×10⁶ cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml detacha-bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentrationof 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×10⁵cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml ofIL-7. Recombinant human IL-10 is added the next day at a finalconcentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherentcells: Seven and fourteen days after the primary induction, the cellsare restimulated with peptide-pulsed adherent cells. The PBMCs arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×10⁶ cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the nonadherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml β₂ microglobulin in 0.25 mlRPMI/5% AB per well for 2 hours at 37° C. Peptide solution from eachwell is aspirated and the wells are washed once with RPMI. Most of themedia is aspirated from the induction cultures (CD8+ cells) and broughtto 0.5 ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 ng/mland recombinant human IL2 is added the next day and again 2-3 days laterat 50 IU/m1 (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a⁵¹Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined ina standard (5 hr) ⁵¹Cr release assay by assaying individual wells at asingle E:T. Peptide-pulsed targets are prepared by incubating the cellswith 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA.Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont,Wilmington, Del.) for 1 hour at 37° C. Labeled target cells areresuspended at 10⁶ per ml and diluted 1:10 with K562 cells at aconcentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:[(cpm of the test sample−cpm of the spontaneous ⁵¹Cr releasesample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeledtargets with 1% Triton X-100 and media alone, respectively. A positiveculture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

In situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates arewashed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTLs (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at400 pg or 1200 pg/100 microliter/well and the plate incubated for twohours at 37° C. The plates are washed and 100 μl of biotinylated mouseanti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at roomtemperature. After washing again, 100 microliter HRP-streptavidin(1:4000) are added and the plates incubated for one hour at roomtemperature. The plates are then washed 6× with wash buffer, 100microliter/well developing solution (TMB 1:1) are added, and the platesallowed to develop for 5-15 minutes. The reaction is stopped with 50microliter/well 1M H₃PO₄ and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flaskcontaining the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×10⁶/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assayor at 1×10⁶/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Thosecultures that demonstrate specific lytic activity against peptide andendogenous targets are selected and 5×10⁴ CD8⁺ cells are added to a T25flask containing the following: 1×10⁶ autologous PBMC per ml which havebeen peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. andirradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10% (v/v) human AB serum,non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine andgentamicin.

Immunogenicity of A2 Supermotif-bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellularassay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patientsbearing a tumor that expresses 161P5C5. Briefly, PBMCs are isolated frompatients, re-stimulated with peptide-pulsed monocytes and assayed forthe ability to recognize peptide-pulsed target cells as well astransfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are alsoevaluated for immunogenicity using methodology analogous for that usedto evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive bindingpeptides identified as set forth herein are confirmed in a manneranalogous to the confirmation of A2-and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc.are also confirmed using similar methodology

Example 15 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase thecross-reactivity of the epitopes. For example, the main anchors ofA2-supermotif-bearing peptides are altered, for example, to introduce apreferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineeredanalog is initially tested for binding to the prototype A2 supertypeallele A*0201, then, if A*0201 binding capacity is maintained, forA2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertypemembers and then analoged to modulate binding affinity to any one (ormore) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screeninganalysis is typically further restricted by the capacity of the parentwild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of5000 nM or less, to three of more A2 supertype alleles. The rationalefor this requirement is that the WT peptides must be presentendogenously in sufficient quantity to be biologically relevant.Analoged peptides have been shown to have increased immunogenicity andcross-reactivity by T cells specific for the parent epitope (see, e.g.,Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc.Natl. Acad. Sci. USA 92:8166, 1995).

In the cellular screening of these peptide analogs, it is important toconfirm that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, target cells that endogenouslyexpress the epitope.

Analoging of HLA-A3 and B7-Supermotif-bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 andA*11 (prototype A3 supertype alleles). Those peptides that demonstrate≦500 nM binding capacity are then confirmed as having A3-supertypecross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3or more B7-supertype alleles can be improved, where possible, to achieveincreased cross-reactive binding or greater binding affinity or bindinghalf life. B7 supermotif-bearing peptides are, for example, engineeredto possess a preferred residue (V, I, L, or F) at the C-terminal primaryanchor position, as demonstrated by Sidney et al. (J. Immunol.157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typicallyin a cellular screening assay. Again, it is generally important todemonstrate that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, targets that endogenously expressthe epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analoged peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with161P5C5-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I.Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 16 Identification and Confirmation of 161P5C5-derived Sequenceswith HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

Selection of HLA-DR-supermotif-bearing Epitopes.

To identify 161P5C5-derived, HLA class II HTL epitopes, a 161P5C5antigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have beendeveloped (Southwood et al., J. Immunol. 160:3363-3373, 1998). Theseprotocols, specific for individual DR molecules, allow the scoring, andranking, of 9-mer core regions. Each protocol not only scores peptidesequences for the presence of DR-supermotif primary anchors (i.e., atposition 1 and position 6) within a 9-mer core, but additionallyevaluates sequences for the presence of secondary anchors. Usingallele-specific selection tables (see, e.g., Southwood et al., ibid.),it has been found that these protocols efficiently select peptidesequences with a high probability of binding a particular DR molecule.Additionally, it has been found that performing these protocols intandem, specifically those for DR1, DR4w4, and DR7, can efficientlyselect DR cross-reactive peptides.

The 161P5C5-derived peptides identified above are tested for theirbinding capacity for various common HLA-DR molecules. All peptides areinitially tested for binding to the DR molecules in the primary panel:DR1, DR4w4, and DR7. Peptides binding at least two of these three DRmolecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, andDR9 molecules in secondary assays. Finally, peptides binding at leasttwo of the four secondary panel DR molecules, and thus cumulatively atleast four of seven different DR molecules, are screened for binding toDR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides bindingat least seven of the ten DR molecules comprising the primary,secondary, and tertiary screening assays are considered cross-reactiveDR binders. 161P5C5-derived peptides found to bind common HLA-DR allelesare of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, andHispanic populations, DR3 binding capacity is a relevant criterion inthe selection of HTL epitopes. Thus, peptides shown to be candidates mayalso be assayed for their DR3 binding capacity. However, in view of thebinding specificity of the DR3 motif, peptides binding only to DR3 canalso be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 161P5C5 antigensare analyzed for sequences carrying one of the two DR3-specific bindingmotifs reported by Geluk, et al. (J. Immunol. 152:5742-5748, 1994). Thecorresponding peptides are then synthesized and confirmed as having theability to bind DR3 with an affinity of 1 μM or better, i.e., less than1 μM. Peptides are found that meet this binding criterion and qualify asHLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccinecompositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the classII motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 17 Immunogenicity of 161P5C5-derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearingepitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous tothe determination of immunogenicity of CTL epitopes, by assessing theability to stimulate HTL responses and/or by using appropriatetransgenic mouse models. Immunogenicity is determined by screening for:1.) in vitro primary induction using normal PBMC or 2.) recall responsesfrom patients who have 161P5C5-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of populationcoverage of a vaccine composition comprised of multiple epitopescomprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA allelesare determined. Gene frequencies for each HLA allele are calculated fromantigen or allele frequencies utilizing the binomial distributionformulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol.45:79-93, 1996). To obtain overall phenotypic frequencies, cumulativegene frequencies are calculated, and the cumulative antigen frequenciesderived by the use of the inverse formula [af=1−(1−Cgf)²].

Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypesis approximately 86% in five major ethnic groups. Coverage may beextended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%. An analogous approach can be used to estimatepopulation coverage achieved with combinations of class II motif-bearingepitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest.100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al.,J. Immunol. 159:1648, 1997) have shown that highly cross-reactivebinding peptides are almost always recognized as epitopes. The use ofhighly cross-reactive binding peptides is an important selectioncriterion in identifying candidate epitopes for inclusion in a vaccinethat is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from theart), an average population coverage is predicted to be greater than 95%in each of five major ethnic populations. The game theory Monte Carlosimulation analysis, which is known in the art (see e.g., Osborne, M. J.and Rubinstein, A. “A course in game theory” MIT Press, 1994), can beused to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 19 CTL Recognition of Endogenously Processed Antigens AfterPriming

This example confirms that CTL induced by native or analoged peptideepitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized withpeptide epitopes, for example HLA-A2 supermotif-bearing epitopes, arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 161P5C5 expression vectors.

The results demonstrate that CTL lines obtained from animals primed withpeptide epitope recognize endogenously synthesized 161P5C5 antigen. Thechoice of transgenic mouse model to be used for such an analysis dependsupon the epitope(s) that are being evaluated. In addition toHLA-A*0201/K^(b) transgenic mice, several other transgenic mouse modelsincluding mice with human A11, which may also be used to evaluate A3epitopes, and B7 alleles have been characterized and others (e.g.,transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 andHLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 20 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenicmice, by use of a 161P5C5-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 161P5C5-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed asdescribed (Alexander et al., J. Immunol. 159:4753-4761, 1997). Forexample, A2/K^(b) mice, which are transgenic for the human HLA A2.1allele and are used to confirm the immunogenicity of HLA-A*0201 motif-or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously(base of the tail) with a 0.1 ml of peptide in Incomplete Freund'sAdjuvant, or if the peptide composition is a lipidated CTL/HTLconjugate, in DMSO/saline, or if the peptide composition is apolypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days afterpriming, splenocytes obtained from these animals are restimulated withsyngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays areJurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g.,Vitiello et al., J. Exp. Med. 173:1007, 1991)

In vitro CTL activation: One week after priming, spleen cells (30×10⁶cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml ofculture medium/T25 flask. After six days, effector cells are harvestedand assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) areincubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10⁴⁵¹Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Crrelease assay. To obtain specific lytic units/10⁶, the lytic units/10⁶obtained in the absence of peptide is subtracted from the lyticunits/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×10⁵ effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be:[(1/50,000)−(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation and are compared to the magnitude of the CTL responseachieved using, for example, CTL epitopes as outlined above in theExample entitled “Confirmation of Immunogenicity.” Analyses similar tothis may be performed to confirm the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in a161P5C5-specific Vaccine

This example illustrates a procedure for selecting peptide epitopes forvaccine compositions of the invention. The peptides in the compositioncan be in the form of a nucleic acid sequence, either single or one ormore sequences (i.e., minigene) that encodes peptide(s), or can besingle and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality ofepitopes for inclusion in a vaccine composition. Each of the followingprinciples is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responsesthat are correlated with 161P5C5 clearance. The number of epitopes useddepends on observations of patients who spontaneously clear 161P5C5. Forexample, if it has been observed that patients who spontaneously clear161P5C5-expressing cells generate an immune response to at least three(3) epitopes from 161P5C5 antigen, then at least three epitopes shouldbe included for HLA class I. A similar rationale is used to determineHLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of1000 nM or less; or HLA Class I peptides with high binding scores fromthe BIMAS web site.

In order to achieve broad coverage of the vaccine through out a diversepopulation, sufficient supermotif bearing peptides, or a sufficientarray of allele-specific motif bearing peptides, are selected to givebroad population coverage. In one embodiment, epitopes are selected toprovide at least 80% population coverage. A Monte Carlo analysis, astatistical evaluation known in the art, can be employed to assessbreadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodessame, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes may be nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9-mer epitopes and one 10-mer epitope can be present in a10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 161P5C5, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered,is safe, efficacious, and elicits an immune response similar inmagnitude to an immune response that controls or clears cells that bearor overexpress 161P5C5.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 161P5C5, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 161P5C5 to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the Ii protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the Ii protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class IImolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified. The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to which it InducesImmunogenicity

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761,1994.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunizedintramuscularly with 100 μof naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a ⁵¹Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-A^(b)-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael,Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med.5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNAminigene encoding the immunogenic peptides including at least one HLA-A2supermotif-bearing peptide. After an incubation period (ranging from 3-9weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinantvaccinia virus expressing the same sequence encoded by the DNA minigene.Control mice are immunized with 100 μg of DNA or recombinant vacciniawithout the minigene sequence, or with DNA encoding the minigene, butwithout the vaccinia boost. After an additional incubation period of twoweeks, splenocytes from the mice are immediately assayed forpeptide-specific activity in an ELISPOT assay. Additionally, splenocytesare stimulated in vitro with the A2-restricted peptide epitopes encodedin the minigene and recombinant vaccinia, then assayed forpeptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 24 Peptide Compositions for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent161P5C5 expression in persons who are at risk for tumors that bear thisantigen. For example, a polyepitopic peptide epitope composition (or anucleic acid comprising the same) containing multiple CTL and HTLepitopes such as those selected in the above Examples, which are alsoselected to target greater than 80% of the population, is administeredto individuals at risk for a 161P5C5-associated tumor.

For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freund's Adjuvant. The dose of peptide for theinitial immunization is from about 1 to about 50,000 μg, generally100-5,000 μg, for a 70 kg patient. The initial administration of vaccineis followed by booster dosages at 4 weeks followed by evaluation of themagnitude of the immune response in the patient, by techniques thatdetermine the presence of epitope-specific CTL populations in a PBMCsample. Additional booster doses are administered as required. Thecomposition is found to be both safe and efficacious as a prophylaxisagainst 161P5C5-associated disease.

Alternatively, a composition typically comprising transfecting agents isused for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native 161P5C5Sequences

A native 161P5C5 polyprotein sequence is analyzed, preferably usingcomputer algorithms defined for each class I and/or class II supermotifor motif, to identify “relatively short” regions of the polyprotein thatcomprise multiple epitopes. The “relatively short” regions arepreferably less in length than an entire native antigen. This relativelyshort sequence that contains multiple distinct or overlapping, “nested”epitopes can be used to generate a minigene construct. The construct isengineered to express the peptide, which corresponds to the nativeprotein sequence. The “relatively short” peptide is generally less than250 amino acids in length, often less than 100 amino acids in length,preferably less than 75 amino acids in length, and more preferably lessthan 50 amino acids in length. The protein sequence of the vaccinecomposition is selected because it has maximal number of epitopescontained within the sequence, i.e., it has a high concentration ofepitopes. As noted herein, epitope motifs may be nested or overlapping(i.e., frame shifted relative to one another). For example, withoverlapping epitopes, two 9-mer epitopes and one 10-mer epitope can bepresent in a 10 amino acid peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopesfrom 161P5C5 antigen and at least one HTL epitope. This polyepitopicnative sequence is administered either as a peptide or as a nucleic acidsequence which encodes the peptide. Alternatively, an analog can be madeof this native sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally, such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup(s) that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 161P5C5, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

Related to this embodiment, computer programs are available in the artwhich can be used to identify in a target sequence, the greatest numberof epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

The 161P5C5 peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 161P5C5 and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from 161P5C5 as well astumor-associated antigens that are often expressed with a target cancerassociated with 161P5C5 expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to 161P5C5.Such an analysis can be performed in a manner described by Ogg et al.,Science 279:2103-2106, 1998. In this Example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) are used for a cross-sectional analysis of, forexample, 161P5C5 HLA-A*0201-specific CTL frequencies from HLAA*0201-positive individuals at different stages of disease or followingimmunization comprising a 161P5C5 peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey et al., N.Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201in this example) and β2-microglobulin are synthesized by means of aprokaryotic expression system. The heavy chain is modified by deletionof the transmembrane-cytosolic tail and COOH-terminal addition of asequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl ofcold phosphate-buffered saline. Tri-color analysis is performed with thetetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. ThePBMCs are incubated with tetramer and antibodies on ice for 30 to 60 minand then washed twice before formaldehyde fixation. Gates are applied tocontain >99.98% of control samples. Controls for the tetramers includeboth A*0201-negative individuals and A*0201-positive non-diseaseddonors. The percentage of cells stained with the tetramer is thendetermined by flow cytometry. The results indicate the number of cellsin the PBMC sample that contain epitope-restricted CTLs, thereby readilyindicating the extent of immune response to the 161P5C5 epitope, andthus the status of exposure to 161P5C5, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses, such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from161P5C5-associated disease or who have been vaccinated with a 161P5C5vaccine.

For example, the class I restricted CTL response of persons who havebeen vaccinated may be analyzed. The vaccine may be any 161P5C5 vaccine.PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. A synthetic peptide comprising an epitope of theinvention is added at 10 μg/ml to each well and HBV core 128-140 epitopeis added at 1 μg/ml to each well as a source of T cell help during thefirst week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with the syntheticpeptide epitope of the invention at 10 μM, and labeled with 100 μCi of⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after whichthey are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Crrelease assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release-spontaneous release)/maximumrelease-spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 161P5C5 or a 161P5C5 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptideof the invention, whole 161P5C5 antigen, or PHA. Cells are routinelyplated in replicates of 4-6 wells for each condition. After seven daysof culture, the medium is removed and replaced with fresh mediumcontaining 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added toeach well and incubation is continued for an additional 18 hours.Cellular DNA is then harvested on glass fiber mats and analyzed for3H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 29 Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials in Patients Expressing 161P5C5

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients having cancer that expresses161P5C5. The main objectives of the trial are to determine an effectivedose and regimen for inducing CTLs in cancer patients that express161P5C5, to establish the safety of inducing a CTL and HTL response inthese patients, and to see to what extent activation of CTLs improvesthe clinical picture of these patients, as manifested, e.g., by thereduction and/or shrinking of lesions. Such a study is designed, forexample, as follows:

The studies are performed in multiple centers. The trial design is anopen-label, uncontrolled, dose escalation protocol wherein the peptidecomposition is administered as a single dose followed six weeks later bya single booster shot of the same dose. The dosages are 50, 500 and5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and represent diverseethnic backgrounds. All of them have a tumor that expresses 161P5C5.

Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 161P5C5-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against161P5C5 is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells(DC)

Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 161P5C5 protein from which the epitopes in thevaccine are derived.

For example, a cocktail of epitope-comprising peptides is administeredex vivo to PBMC, or isolated DC therefrom. A pharmaceutical tofacilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

As appreciated clinically, and readily determined by one of skill basedon clinical outcomes, the number of DC reinfused into the patient canvary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2-50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patientswithout purification of the DC. For example, PBMC generated aftertreatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

Ex vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 161P5C5 antigens can beinduced by incubating, in tissue culture, the patient's, or geneticallycompatible, CTL or HTL precursor cells together with a source of APC,such as DC, and immunogenic peptides. After an appropriate incubationtime (typically about 7-28 days), in which the precursor cells areactivated and expanded into effector cells, the cells are infused intothe patient, where they will destroy (CTL) or facilitate destruction(HTL) of their specific target cells, i.e., tumor cells.

Example 33 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides isto elute them from cells bearing defined MHC molecules. For example, EBVtransformed B cell lines used for tissue typing have been extensivelycharacterized to determine which HLA molecules they express. In certaincases these cells express only a single type of HLA molecule. Thesecells can be transfected with nucleic acids that express the antigen ofinterest, e.g. 161P5C5. Peptides produced by endogenous antigenprocessing of peptides produced as a result of transfection will thenbind to HLA molecules within the cell and be transported and displayedon the cell's surface. Peptides are then eluted from the HLA moleculesby exposure to mild acid conditions and their amino acid sequencedetermined, e.g., by mass spectral analysis (e.g., Kubo et al., J.Immunol. 152:3913, 1994). Because the majority of peptides that bind aparticular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA moleculescan be transfected with an expression construct encoding a single HLAallele. These cells can then be used as described, i.e., they can thenbe transfected with nucleic acids that encode 161P5C5 to isolatepeptides corresponding to 161P5C5 that have been presented on the cellsurface. Peptides obtained from such an analysis will bear motif(s) thatcorrespond to binding to the single HLA allele that is expressed in thecell.

As appreciated by one in the art, one can perform a similar analysis ona cell bearing more than one HLA allele and subsequently determinepeptides specific for each HLA allele expressed. Moreover, one of skillwould also recognize that means other than transfection, such as loadingwith a protein antigen, can be used to provide a source of antigen tothe cell.

Example 34 Complementary Polynucleotides

Sequences complementary to the 161P5C5-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 161P5C5. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 161P5C5. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to a 161P5C5-encodingtranscript.

Example 35 Purification of Naturally-occurring or Recombinant 161P5C5Using 161P5C5-Specific Antibodies

Naturally occurring or recombinant 161P5C5 is substantially purified byimmunoaffinity chromatography using antibodies specific for 161P5C5. Animmunoaffinity column is constructed by covalently coupling anti-161P5C5antibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing 161P5C5 are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of 161P5C5 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/161P5C5 binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 36 Identification of Molecules which Interact with 161P5C5

161P5C5, or biologically active fragments thereof, are labeled with 1211 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled 161P5C5, washed, and anywells with labeled 161P5C5 complex are assayed. Data obtained usingdifferent concentrations of 161P5C5 are used to calculate values for thenumber, affinity, and association of 161P5C5 with the candidatemolecules.

Example 37 In Vivo Assay for 161P5C5 Tumor Growth Promotion

The effect of the 161P5C5 protein on tumor cell growth is evaluated invivo by evaluating tumor development and growth of cells expressing orlacking 161P5C5. For example, SCID mice are injected subcutaneously oneach flank with 1×10⁶ of either 3T3, bladder, kidney or ovary cancercell lines (e.g. UM-UC3, J82, PA-1, CaOv3, CaKi1 or 769P cells)containing tkNeo empty vector or 161P5C5. At least two strategies may beused: (1) Constitutive 161P5C5 expression under regulation of a promotersuch as a constitutive promoter obtained from the genomes of virusessuch as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul.1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, aviansarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus andSimian Virus 40 (SV40), or from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, provided suchpromoters are compatible with the host cell systems, and (2) Regulatedexpression under control of an inducible vector system, such asecdysone, tetracycline, etc., provided such promoters are compatiblewith the host cell systems. Tumor volume is then monitored by calipermeasurement at the appearance of palpable tumors and followed over timeto determine if 161P5C5-expressing cells grow at a faster rate andwhether tumors produced by 161P5C5-expressing cells demonstratecharacteristics of altered aggressiveness (e.g. enhanced metastasis,vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1×10⁵ of the same cellsorthotopically to determine if 161P5C5 has an effect on local growth inthe bladder, kidney or ovary, and whether 161P5C5 affects the ability ofthe cells to metastasize, specifically to lymph nodes, adrenal, liverand bone (Miki T, et al., Oncol Res. 2001; 12:209; Fu X, et al., Int JCancer. 1991, 49:938; Kiguchi Ket al, Clin Exp Metastasis. 1998,16:751).

The assay is also useful to determine the 161P5C5 inhibitory effect ofcandidate therapeutic compositions, such as for example, 161P5C5intrabodies, 161P5C5 antisense molecules and ribozymes.

Example 38 161P5C5 Monoclonal Antibody-mediated Inhibition of Bladder,Kidney and Ovarian Tumors In Vivo

The significant expression of 161P5C5 in cancer tissues, together withits restrictive expression in normal tissues makes 161P5C5 a good targetfor antibody therapy. Similarly, 161P5C5 is a target for T cell-basedimmunotherapy. Thus, the therapeutic efficacy of anti-161P5C5 mAbs inhuman bladder cancer xenograft mouse models is evaluated by usingrecombinant cell lines such as UM-UC3-161P5C5, J82-161P5C5, and3T3-161P5C5 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1):p. 16-23). Similarly, anti-161P5C5 mAbs are evaluated in human kidneyand ovarian cancer xenograft models using recombinant cell lines such asCaKi1-161P5C5 and PA1-161P5C5.

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in a mouse orthotopic bladder cancer xenograft model, a mousekidney cancer xenograft model and a mouse ovarian cancer xenograftmodel. The antibodies can be unconjugated, as discussed in this Example,or can be conjugated to a therapeutic modality, as appreciated in theart. Anti-161P5C5 mAbs inhibit formation of kidney, ovarian and bladderxenografts. Anti-161P5C5 mAbs also retard the growth of establishedorthotopic tumors and prolonged survival of tumor-bearing mice. Theseresults indicate the utility of anti-161P5C5 mAbs in the treatment oflocal and advanced stages of ovarian, kidney and bladder cancer. (See,e.g., Saffran, D., et al., PNAS (2001) 10:1073-1078.)

Administration of the anti-161P5C5 mAbs led to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 161P5C5 is anattractive target for immunotherapy and demonstrate the therapeuticpotential of anti-161P5C5 mAbs for the treatment of local and metastaticcancer. This example demonstrates that unconjugated 161P5C5 monoclonalantibodies are effective to inhibit the growth of human bladder, kidneyand ovarian tumor xenografts grown in SCID mice; accordingly acombination of such efficacious monoclonal antibodies is also effective.

Tumor inhibition using multiple unconjugated 161P5C5 mAbs

Materials and Methods

161P5C5 Monoclonal Antibodies:

Monoclonal antibodies are raised against 161P5C5 as described in theExample entitled “Generation of 161P5C5 Monoclonal Antibodies (mAbs).”The antibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation for their capacity to bind 161P5C5. Epitope mappingdata for the anti-161P5C5 mAbs, as determined by ELISA and Westernanalysis, recognize epitopes on the 161P5C5 protein. Immunohistochemicalanalysis of prostate cancer tissues and cells with these antibodies isperformed.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofUM-UC3, J82, CaKi1, 769P, CaOv1 or PA1 tumor xenografts.

Cell Lines

The bladder, kidney and ovary carcinoma cell lines, UM-UC3, J82, CaKi1,769P, CaOv1 and PA1 as well as the fibroblast line NIH 3T3 (AmericanType Culture Collection) are maintained in DMEM supplemented withL-glutamine and 10% FBS.

A UM-UC3-161P5C5, J82-161P5C5, CaKi1-161P5C5, 769P-161P5C5,CaOv1-161P5C5, PA1-161P5C5 and 3T3-161P5C5 cell populations aregenerated by retroviral gene transfer as described in Hubert, R. S., etal., Proc Natl Acad Sci USA, 1999. 96(25): 14523.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ cancercells mixed at a 1:1 dilution with Matrigel (Collaborative Research) inthe right flank of male SCID mice. To test antibody efficacy on tumorformation, i.p. antibody injections are started on the same day astumor-cell injections. As a control, mice are injected with eitherpurified mouse IgG (ICN) or PBS; or a purified monoclonal antibody thatrecognizes an irrelevant antigen not expressed in human cells. Inpreliminary studies, no difference is found between mouse IgG or PBS ontumor growth. Tumor sizes are determined by caliper measurements, andthe tumor volume is calculated as length×width×height. Mice with s.c.tumors greater than 1.5 cm in diameter are sacrificed.

Orthotopic injections are performed under anesthesia by usingketamine/xylazine. For bladder orthotopic studies, an incision is madethrough the abdomen to expose the bladder, and tumor cells (5×10⁵) mixedwith Matrigel are injected into the bladder wall in a 10-μl volume. Tomonitor tumor growth, mice are palpated and blood is collected on aweekly basis to measure BTA levels. For kidney and ovary orthopoticmodels, an incision is made through the abdominal muscles to expose thekidney or the ovary. Tumor cells mixed with Matrigel are injected underthe kidney capsule or into the ovary in a 10-μl volume (Yoshida Y, etal., Anticancer Res. 1998, 18:327; Ahn, et al., Tumour Biol. 2001,22:146). To monitor tumor growth, blood is collected on a weekly basismeasuring G250 and SM047 levels. The mice are segregated into groups forthe appropriate treatments, with anti-161P5C5 or control mAbs beinginjected i.p.

Anti-161P5C5 mAbs Inhibit Growth of 161P5C5-Expressing Xenograft-CancerTumors

The effect of anti-161P5C5 mAbs on tumor formation is tested on thegrowth and progression of bladder, kidney and ovarian cancer xenograftsusing UC3-161P5C5, J82-161P5C5, CaKi1-161P5C5, 769P-161P5C5,CaOv1-161P5C5 and PA1-161P5C5 orthotopic models. As compared with thes.c. tumor model, the orthotopic model, which requires injection oftumor cells directly in the mouse bladder, kidney and ovary,respectively, results in a local tumor growth, development of metastasisin distal sites, deterioration of mouse health, and subsequent death(Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992.52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). Thefeatures make the orthotopic model more representative of human diseaseprogression and allowed us to follow the therapeutic effect of mAbs onclinically relevant end points.

Accordingly, tumor cells are injected into the mouse bladder, kidney orovary, and 2 days later, the mice are segregated into two groups andtreated with either: a) 200-500 μg, of anti-161P5C5 Ab, or b) PBS threetimes per week for two to five weeks.

A major advantage of the orthotopic cancer models is the ability tostudy the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against a tumor-specific cell-surface proteinsuch as anti-CK20 for bladder cancer, anti-G250 for kidney cancer andSM047 antibody for ovarian cancer models (Lin S, et al., Cancer DetectPrey. 2001; 25:202; McCluggage W, et al., Histopathol 2001, 38:542).

Mice bearing established orthotopic tumors are administered 1000 μginjections of either anti-161P5C5 mAb or PBS over a 4-week period. Micein both groups are allowed to establish a high tumor burden, to ensure ahigh frequency of metastasis formation in mouse lungs. Mice then arekilled and their bladders, livers, bone and lungs are analyzed for thepresence of tumor cells by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-161P5C5antibodies on initiation and progression of prostate and kidney cancerin xenograft mouse models. Anti-161P5C5 antibodies inhibit tumorformation of tumors as well as retarding the growth of alreadyestablished tumors and prolong the survival of treated mice. Moreover,anti-161P5C5 mAbs demonstrate a dramatic inhibitory effect on the spreadof local bladder, kidney and ovarian tumor to distal sites, even in thepresence of a large tumor burden. Thus, anti-161P5C5 mAbs areefficacious on major clinically relevant end points (tumor growth),prolongation of survival, and health.

Example 39 Therapeutic and Diagnostic Use of Anti-161P5C5 Antibodies inHumans

Anti-161P5C5 monoclonal antibodies are safely and effectively used fordiagnostic, prophylactic, prognostic and/or therapeutic purposes inhumans. Western blot and immunohistochemical analysis of cancer tissuesand cancer xenografts with anti-161P5C5 mAb show strong extensivestaining in carcinoma but significantly lower or undetectable levels innormal tissues. Detection of 161P5C5 in carcinoma and in metastaticdisease demonstrates the usefulness of the mAb as a diagnostic and/orprognostic indicator. Anti-161P5C5 antibodies are therefore used indiagnostic applications such as immunohistochemistry of kidney biopsyspecimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-161P5C5 mAb specifically binds tocarcinoma cells. Thus, anti-161P5C5 antibodies are used in diagnosticwhole body imaging applications, such as radioimmunoscintigraphy andradioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res20(2A):925-948 (2000)) for the detection of localized and metastaticcancers that exhibit expression of 161P5C5. Shedding or release of anextracellular domain of 161P5C5 into the extracellular milieu, such asthat seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology27:563-568 (1998)), allows diagnostic detection of 161P5C5 byanti-161P5C5 antibodies in serum and/or urine samples from suspectpatients.

Anti-161P5C5 antibodies that specifically bind 161P5C5 are used intherapeutic applications for the treatment of cancers that express161P5C5. Anti-161P5C5 antibodies are used as an unconjugated modalityand as conjugated form in which the antibodies are attached to one ofvarious therapeutic or imaging modalities well known in the art, such asa prodrugs, enzymes or radioisotopes. In preclinical studies,unconjugated and conjugated anti-161P5C5 antibodies are tested forefficacy of tumor prevention and growth inhibition in the SCID mousecancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6,(see, e.g., the Example entitled “161P5C5 Monoclonal Antibody-mediatedInhibition of Bladder, Kidney and Ovarian Tumors In Vivo”). Conjugatedand unconjugated anti-161P5C5 antibodies are used as a therapeuticmodality in human clinical trials either alone or in combination withother treatments as described in following Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas Through Use of Human Anti-161P5C5 Antibodies In vivo

Antibodies are used in accordance with the present invention whichrecognize an epitope on 161P5C5, and are used in the treatment ofcertain tumors such as those listed in Table I. Based upon a number offactors, including 161P5C5 expression levels, tumors such as thoselisted in Table I are presently preferred indications. In connectionwith each of these indications, three clinical approaches aresuccessfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated withanti-161P5C5 antibodies in combination with a chemotherapeutic orantineoplastic agent and/or radiation therapy. Primary cancer targets,such as those listed in Table I, are treated under standard protocols bythe addition anti-161P5C5 antibodies to standard first and second linetherapy. Protocol designs address effectiveness as assessed by reductionin tumor mass as well as the ability to reduce usual doses of standardchemotherapy. These dosage reductions allow additional and/or prolongedtherapy by reducing dose-related toxicity of the chemotherapeutic agent.Anti-161P5C5 antibodies are utilized in several adjunctive clinicaltrials in combination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

II.) Monotherapy: In connection with the use of the anti-161P5C5antibodies in monotherapy of tumors, the antibodies are administered topatients without a chemotherapeutic or antineoplastic agent. In oneembodiment, monotherapy is conducted clinically in end stage cancerpatients with extensive metastatic disease. Patients show some diseasestabilization. Trials demonstrate an effect in refractory patients withcancerous tumors.

III.) Imaging Agent: Through binding a radionuclide (e.g., iodine oryttrium (I¹³¹, Y⁹⁰) to anti-161P5C5 antibodies, the radiolabeledantibodies are utilized as a diagnostic and/or imaging agent. In such arole, the labeled antibodies localize to both solid tumors, as well as,metastatic lesions of cells expressing 161P5C5. In connection with theuse of the anti-161P5C5 antibodies as imaging agents, the antibodies areused as an adjunct to surgical treatment of solid tumors, as both apre-surgical screen as well as a post-operative follow-up to determinewhat tumor remains and/or returns. In one embodiment, a (¹¹¹In)-161P5C5antibody is used as an imaging agent in a Phase I human clinical trialin patients having a carcinoma that expresses 161P5C5 (by analogy see,e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients arefollowed with standard anterior and posterior gamma camera. The resultsindicate that primary lesions and metastatic lesions are identified

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-161P5C5 antibodies can beadministered with doses in the range of 5 to 400 mg/m², with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-161P5C5 antibodies relative to the affinity of a known antibody forits target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-161P5C5 antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-161P5C5 antibodies can be lower, perhaps in the range of 50to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposedto the conventional measurement of dose in mg/kg, is a measurement basedon surface area and is a convenient dosing measurement that is designedto include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery ofanti-161P5C5 antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner, certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allows for a high dose of antibody at the site of atumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-161P5C5antibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-161P5C5antibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is 161P5C5 expression levels intheir tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 161P5C5.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-161P5C5 antibodies are found to be safe upon humanadministration.

Example 41 Human Clinical Trial Adjunctive Therapy with HumanAnti-161P5C5 Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of sixintravenous doses of a human anti-161P5C5 antibody in connection withthe treatment of a solid tumor, e.g., a cancer of a tissue listed inTable I. In the study, the safety of single doses of anti-161P5C5antibodies when utilized as an adjunctive therapy to an antineoplasticor chemotherapeutic agent, such as cisplatin, topotecan, doxorubicin,adriamycin, taxol, or the like, is assessed. The trial design includesdelivery of six single doses of an anti-161P5C5 antibody with dosage ofantibody escalating from approximately about 25 mg/m² to about 275 mg/m²over the course of the treatment in accordance with the followingschedule:

Day Day Day Day Day 0 Day 7 14 21 28 35 mAb Dose 25 mg/m² 75 mg/m² 125mg/m² 175 mg/m² 225 mg/m² 275 mg/m² Chemotherapy + + + + + + (standarddose)

Patients are closely followed for one-week following each administrationof antibody and chemotherapy. In particular, patients are assessed forthe safety concerns mentioned above: (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the human antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 161P5C5.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Patients are also assessed for clinical outcome, andparticularly reduction in tumor mass as evidenced by MRI or otherimaging.

The anti-161P5C5 antibodies are demonstrated to be safe and efficacious,Phase II trials confirm the efficacy and refine optimum dosing.

Example 42 Human Clinical Trial: Monotherapy with Human Anti-161P5C5Antibody

Anti-161P5C5 antibodies are safe in connection with the above-discussedadjunctive trial, a Phase II human clinical trial confirms the efficacyand optimum dosing for monotherapy. Such trial is accomplished, andentails the same safety and outcome analyses, to the above-describedadjunctive trial with the exception being that patients do not receivechemotherapy concurrently with the receipt of doses of anti-161P5C5antibodies.

Example 43 Human Clinical Trial: Diagnostic Imaging with Anti-161P5C5Antibody

Once again, as the adjunctive therapy discussed above is safe within thesafety criteria discussed above, a human clinical trial is conductedconcerning the use of anti-161P5C5 antibodies as a diagnostic imagingagent. The protocol is designed in a substantially similar manner tothose described in the art, such as in Divgi et al. J. Natl. CancerInst. 83:97-104 (1991). The antibodies are found to be both safe andefficacious when used as a diagnostic modality.

Example 44 Homology Comparison of 161P5C5 to Known Sequences

The 161P5C5 gene exhibits some homology to a previously cloned gene,namely the sea urchin bindin fertilization specificity protein (gi114993). The 161P5C5 shows 38% identity and 49% homology to the bindinfertilization specificity protein over 34 amino acids; see FIG. 4C. Inaddition, the 161P5C5 protein shows homology to protoporphyrinogenoxidase (gi 15836421), exhibiting 38% identity and 62% homology over 49amino acids; see FIG. 4D. Three different variants of 161P5C5 have beenidentified, each differing from each other by one nucleotide resultingin one amino acid change, indicating that 161P5C5-V.1, -V.2 and -V.3 areSNPs. The 161P5C5 protein consists of 71 amino acids, with calculatedmolecular weight of 8.6 kDa, and pI of 7.9. 161P5C5 is predicted to bean intracellular protein, which localizes to the endoplasmic reticulumand cytosol. 161P5C5 can also localize to the cytoskeleton and lysosome.Motif analysis revealed the presence of WHEP-TRS domain at amino acids13-28. See Table XXI for analysis of motifs and physical properties of161P5C5.

A WHEP-TRS domain is a 46 amino acid long conserved domain often foundin eukaryotic aminoacyl-transfer RNA synthetases. This domain mediatesprotein association and the interaction of .tRNA-synthetases intomultienzyme complexes (Cerini C, et al., EMBO J 1991, 10:4267). Thepresence of a WHEP-TRS domain indicates that 161P5C5 participates inprotein-protein interactions. This is further supported by the homologyof 161P5C5 to bindin fertilization specificity protein. Bindin proteinis an adhesive protein that participates in egg fertilization bymediating the interaction of sperm and egg (Lopez A, et al., Dev Biol1993, 156: 24; Miraglia S , Glabe C. Biochim Biophys Acta 1993,11454:191).

In addition to its homology to bindin, the 161P5C5 protein showshomology to protoporphyrinogen oxidase. Protoporphyrinogen oxidase playsan important role in heme biosynthesis. Mutations in theprotoporphyrinogen oxidase gene and the resulting reduction in enzymeactivity have been associated with the inherited disorder known asporphyria variegate (Dailey H, Dailey T, Cell Mol Biol. 1997, 43:67;Frank J, et al., J. Invest. Dermatol 1998, 110:449). The relationship ofprotoporphyrinogen oxidase with tumor development is not wellestablished. However, two publications have reported its associationwith hepatocellular carcinoma (Germanaud J, et al., Scand. J.Gasrtoenterol 1994, 29:671).

The presence of a WHEP-TRS motif and its homology to protein-proteininteraction bindin protein, along with its homology toprotoporphyrinogen oxidase, indicate that 161P5C5 regulates proteininteractions and participates in the process of tumor formation andprogression. By way of its protein interaction domain, 161P5C5 functionsin regulating signal transduction in mammalian cells, thereby regulatinggene expression and cellular outcomes, including cell proliferation,survival, adhesion, etc., all of which have a direct effect on tumorgrowth and progression.

Accordingly, when 161P5C5 functions as a regulator of proteininteractions, cell growth, tumor formation, or cell signaling, 161P5C5is used for therapeutic, diagnostic, prognostic and/or preventativepurposes. In addition, when a molecule, such as a variant or SNP of161P5C5 is expressed in cancerous tissues, such as those listed in TableI, they are used for therapeutic, diagnostic, prognostic and/orpreventative purposes.

Example 45 Regulation of Transcription

The localization of 161P5C5, coupled to the presence of proteininteraction domains within its sequence, indicate that 161P5C5 modulatesthe transcriptional regulation of eukaryotic genes. Regulation of geneexpression is confirmed, e.g., by studying gene expression in cellsexpressing or lacking 161P5C5. For this purpose, two types ofexperiments are performed.

In the first set of experiments, RNA from parental and161P5C5-expressing cells are extracted and hybridized to commerciallyavailable gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer.2000. 83:246). Resting cells as well as cells treated with FBS, androgenor growth factors are compared. Differentially expressed genes areidentified in accordance with procedures known in the art. Thedifferentially expressed genes are then mapped to biological pathways(Chen K et al. Thyroid. 2001. 11:41.).

In the second set of experiments, specific transcriptional pathwayactivation is evaluated using commercially available (Stratagene)luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc,ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters containconsensus binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways, andrepresent a good tool to ascertain pathway activation and screen forpositive and negative modulators of pathway activation.

Thus, 161P5C5 plays a role in gene regulation, and it is used as atarget for diagnostic, prognostic, preventative and/or therapeuticpurposes.

Example 46 Identification and Confirmation of Potential SignalTransduction Pathways

Many mammalian proteins have been reported to interact with signalingmolecules and to participate in regulating signaling pathways. (JNeurochem. 2001; 76:217-223). In particular, protein interaction motifshave been instrumental in inducing kinase activation, recruitment ofproteins and complex formation (Samelson L. Annu Rev Immunol. 2002;20:371). Based on the presence of a protein interaction motif 161P5C5regulates signaling pathways important for cell growth and invasion.Using immunoprecipitation and Western blotting techniques, proteins areidentified that associate with 161P5C5 and mediate signaling events.Several pathways known to play a role in cancer biology can be regulatedby 161P5C5, including phospholipid pathways such as PI3K, AKT, etc,adhesion and migration pathways, including FAK, Rho, Rac-1, β-catenin,etc, as well as mitogenic/survival cascades such as ERK, p38, etc (CellGrowth Differ. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,19:3003, J. Cell Biol. 1997, 138:913.).

To confirm that 161P5C5 directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing individual genes.These transcriptional reporters contain consensus-binding sites forknown transcription factors that lie downstream of well-characterizedsignal transduction pathways. The reporters and examples of theseassociated transcription factors, signal transduction pathways, andactivation stimuli are listed below.

-   -   NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress    -   SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation    -   AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress    -   ARE-luc, androgen receptor; steroids/MAPK;        growth/differentiation/apoptosis    -   p53-luc, p53; SAPK; growth/differentiation/apoptosis    -   CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress    -   TCF-luc, TCF/Lef; β-catenin, Adhesion/invasion

Gene-mediated effects can be assayed in cells showing mRNA expression.Luciferase reporter plasmids can be introduced by lipid-mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer. Moreover, the 161P5C5 protein contains atleast one phosphorylation site (Table XX), indicating its associationwith at least one specific signaling cascade.

Signaling pathways activated by 161P5C5 are mapped and used for theidentification and validation of therapeutic targets. When 161P5C5 isinvolved in cell signaling, it is used as target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 47 Involvement in Tumor Progression

Based on the role of protoporphyrinogen oxidase in tumor formation(Germanaud J, above), the 161P5C5 gene can contribute to tumorinitiation and progression. The role of 161P5C5 in tumor growth isconfirmed in a variety of primary and transfected cell lines includingbladder, kidney and ovary cell lines, as well as NIH 3T3 cellsengineered to stably express 161P5C5. Parental cells lacking 161P5C5 andcells expressing 161P5C5 are evaluated for cell growth using awell-documented proliferation assay (Fraser S P, Grimes J A, Djamgoz MB. Prostate. 2000; 44:61, Johnson D E, Ochieng J, Evans S L. AnticancerDrugs. 1996, 7:288).

To confirm the role of 161P5C5 in the transformation process, its effectin colony forming assays is investigated. Parental NIH-3T3 cells lacking161P5C5 are compared to NIH-3T3 cells expressing 161P5C5, using a softagar assay under stringent and more permissive conditions (Song Z. etal. Cancer Res. 2000; 60:6730).

To confirm the role of 161P5C5 in invasion and metastasis of cancercells, a well-established assay is used, e.g., a Transwell Insert Systemassay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Control cells,including bladder, ovary and kidney cell lines lacking 161P5C5 arecompared to cells expressing 161P5C5. Cells are loaded with thefluorescent dye, calcein, and plated in the top well of the Transwellinsert coated with a basement membrane analog. Invasion is determined byfluorescence of cells in the lower chamber relative to the fluorescenceof the entire cell population.

161P5C5 can also play a role in cell cycle and apoptosis. Parental cellsand cells expressing 161P5C5 are compared for differences in cell cycleregulation using a well-established BrdU assay (Abdel-Malek Z A. J CellPhysiol. 1988, 136:247). In short, cells are grown under both optimal(full serum) and limiting (low serum) conditions are labeled with BrdUand stained with anti-BrdU Ab and propidium iodide. Cells are analyzedfor entry into the G1, S, and G2M phases of the cell cycle.Alternatively, the effect of stress on apoptosis is evaluated in controlparental cells and cells expressing 161P5C5, including normal and tumorbladder, kidney and ovary cells. Engineered and parental cells aretreated with various chemotherapeutic agents, such as etoposide, taxol,etc, and protein synthesis inhibitors, such as cycloheximide. Cells arestained with annexin V-FITC and cell death is measured by FACS analysis.The modulation of cell death by 161P5C5 can play a critical role inregulating tumor progression and tumor load.

When 161P5C5 plays a role in cell growth, transformation, invasion orapoptosis, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

Example 48 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary fortumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J.Endocrinology. 1998 139:441). Based on the effect of phsophodieseteraseinhibitors on endothelial cells, 161P5C5 plays a role in angiogenesis(DeFouw L, et al., Microvasc Res 2001, 62:263). Several assays have beendeveloped to measure angiogenesis in vitro and in vivo, such as thetissue culture assays endothelial cell tube formation and endothelialcell proliferation. Using these assays as well as in vitroneo-vascularization, the role of 161P5C5 in angiogenesis, enhancement orinhibition, is confirmed.

For example, endothelial cells engineered to express 161P5C5 areevaluated using tube formation and proliferation assays. The effect of161P5C5 is also confirmed in animal models in vivo. For example, cellseither expressing or lacking 161P5C5 are implanted subcutaneously inimmunocompromised mice. Endothelial cell migration and angiogenesis areevaluated 5-15 days later using immunohistochemistry techniques. When161P5C5 affects angiogenesis, it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes

Example 49 Involvement in Protein-Protein Interactions

WHEP-TRP motifs have been shown to mediate interaction with otherproteins, resulting in the formation of a multi-protein complex (CeriniC, et al., EMBO J 1991, 10:4267). Using immunoprecipitation techniquesas well as two yeast hybrid systems, proteins are identified thatassociate with 161P5C5. Immunoprecipitates from cells expressing 161P5C5and cells lacking 161P5C5 are compared for specific protein-proteinassociations.

Studies are performed to confirm the extent of association of 161P5C5with effector molecules, such as nuclear proteins, transcriptionfactors, kinases, phosphates, etc. Studies comparing 161P5C5 positiveand 161P5C5 negative cells as well as studies comparingunstimulated/resting cells and cells treated with epithelial cellactivators, such as cytokines, growth factors and anti-integrin Abreveal unique interactions.

In addition, protein-protein interactions are confirmed using two yeasthybrid methodology (Curr Opin Chem Biol. 1999, 3:64). A vector carryinga library of proteins fused to the activation domain of a transcriptionfactor is introduced into yeast expressing a 161P5C5-DNA-binding domainfusion protein and a reporter construct. Protein-protein interaction isdetected by colorimetric reporter activity. Specific association witheffector molecules and transcription factors indicates the mode ofaction of 161P5C5, and thus identifies therapeutic, prognostic,preventative and/or diagnostic targets for cancer. This and similarassays are also used to identify and screen for small molecules thatinteract with 161P5C5.

When 161P5C5 associates with proteins and small molecules, 161P5C5 andthese proteins and small molecules are used for diagnostic, prognostic,preventative and/or therapeutic purposes.

Throughout this application, various website data content, publications,patent applications and patents are referenced.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

TABLE I Tissues that Express 161P5C5 When Malignant Bladder Kidney LungBreast Ovary

TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

TABLE III Amino Acid Substitution Matrix Adapted from the GCG Software9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).The higher the value, the more likely a substitution is found inrelated, natural proteins. A C D E F G H I K L M N P Q R S T V W Y . 4 0−2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2 −3 −3 −1−3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −20 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0−3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2−3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3−3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1 −2 −1 −1 −2 −4−3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4−3 −1 V 11 2 W 7 Y

TABLE IV (A) HLA Class I Supermotifs/Motifs POSITION POSITION POSITION2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary Anchor) SUPERMOTIFS A1 TI LVMS (SEQ ID NO: 57) FWY A2 LIVM ATQ (SEQ ID NO: 58) IVMATL (SEQ ID NO: 59) A3 VSMA TLI (SEQ ID NO: 60) RK A24 YFWIVLMT (SEQ ID NO: 61) FI YWLM (SEQ ID NO: 62) B7 P VILFMWYA (SEQ ID NO: 63) B27 RHK FYL WMIVA (SEQ ID NO: 64) B44 E DFWYLIMVA (SEQ ID NO: 65) B58 ATS FWY LIVMA (SEQ ID NO: 66) B62 QLIVMP (SEQ ID NO: 67) FWYMIVLA (SEQ ID NO: 68) MOTIFS A1 TSM Y A1 DEAS (SEQ ID NO: 180) Y A2.1 LM VQIAT (SEQ ID NO: 69) VLIMAT (SEQ ID NO: 70) A3 LMVISATF CGD (SEQ ID KYR HFA (SEQ ID NO: 72)NO: 71) A11 VTMLISAGN CDF (SEQ ID K RYH (SEQ ID NO: 74) NO: 73) A24 YFWM (SEQ ID NO: 75) FLIW (SEQ ID NO: 76) A*3101 MVT ALIS (SEQ ID NO: 77)R K A*3301 MVALF IST (SEQ ID NO: 78) RK A*6801 AVT MSLI (SEQ ID NO: 79)RK B*0702 P LMF WYAIV (SEQ ID NO: 80) B*3501 P LMFWY IVA (SEQ ID NO: 81)B51 P LIVF WYAM (SEQ ID NO: 82) B*5301 P IMFWY ALV (SEQ ID NO: 83)B*5401 P ATIV LMFWY (SEQ ID NO: 84) Bolded residues are preferred,italicized residues are less preferred: A peptide is consideredmotif-bearing if it has primary anchors at each primary anchor positionfor a motif or supermotif as specified in the above table.

TABLE IV (B) HLA Class II Supermotif 1 6 9 W, F, Y, V, .I, L A, V, I, L,P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C) HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1° anchor 67 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious(SEQ ID NO: 85) W (SEQ ID NO: 86) R WDE DR1 preferred MFLIVWY PAMQVMATSPLIC M AVM deleterious (SEQ ID NO: 87) C CH FD CWD (SEQ ID NO: 89)GDE D (SEQ ID NO: 88) DR7 preferred MFLIVWY M W A IVMSACTPL M IVdeleterious (SEQ ID NO: 90) C G (SEQ ID NO: 91) GRD N G DR3 MOTIFS 1°anchor 1 2 3 1° anchor 4 5 1° anchor 6 motif a LIVMFY D preferred(SEQ ID NO: 92) motif b LIVMFAY DNQEST (SEQ KRH preferred(SEQ ID NO: 93) ID NO: 94) DR MFLIVWY (SEQ VMSTACPLI SupermotifID NO: 95) (SEQ ID NO: 96) Italicized residues indicate less preferredor “tolerated” residues

TABLE IV (D) HLA Class I Supermotifs SUPER- MOTIFS Position: 1 2 3 4 5 67 8 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY (SEQ ID NO: 97) A2 1°Anchor 1° Anchor LIVMATQ LIVMAT (SEQ ID NO: 98) (SEQ ID NO: 99) A3preferred 1° Anchor YFW YFW YFW P 1° Anchor VSMATLI (4/5) (3/5) (4/5)(4/5) RK (SEQ ID NO: 100 deleterious DE (3/5); P (5/5) DE (4/5) A24 1°Anchor 1° Anchor YFWIVLMT FIYWLM (SEQ ID NO: 101) (SEQ ID NO: 102) B7preferred FWY (5/5) LIVM (3/5) 1° Anchor FWY FWY 1° Anchor VILFMWYA(SEQ ID NO: 103) P (4/5) (3/5) (SEQ ID NO: 104) deleteriousDE (3/5); P(5/5); G(4/5); DE G QN DE A(3/5); QN(3/5) (3/5) (4/5) (4/5)(4/5) B27 1° Anchor 1°Anchor RHK FYLWMIVA (SEQ ID NO: 105) B44 1° Anchor1° Anchor ED FWYLIMVA (SEQ ID NO: 106) B58 1° Anchor 1° Anchor ATSFWYLIVMA (SEQ ID NO: 107) B62 1° Anchor 1° Anchor QLIVMPFWYMIVLA (SEQ ID (SEQ ID NO: 108) NO: 109) Italicized residues indicateless preferred or “tolerated” residues

TABLE IV (E) HLA Class I Motifs 9 or C- C- Position: 1 2 3 4 5 6 7 8terminus terminus A1 preferred GFYW 1° Anchor DEA YFW P DEQN YFW 1°Anchor 9-mer (SEQ ID STM (SEQ ID NO: 111) Y NO: 110) deleterious DERHKLIVMP A G A (SEQ ID NO: 112) A1 preferred GRHK ASTCLIVM 1° AnchorGSTC ASTC LIVM DE 1° Anchor 9-mer (SEQ ID (SEQ ID DEAS (SEQ ID (SEQ ID(SEQ ID NO: 118) Y NO: 113) NO: 114) (SEQ ID NO: 115) NO: 116 NO: 117)deleterious A RHKDEPY DE PQN RHK PG GP (SEQ ID NO: 119) FW A1 preferredYFW 1° Anchor DEAQN A YFWQN PASTC GDE P 1° Anchor 10-mer STM(SEQ ID NO: 120) (SEQ ID (SEQ ID NO: 122) Y NO: 121) deleterious GPRHKGLIVM DE RHK QNA RHKYFW RHK A (SEQ ID NO: 123) (SEQ ID NO: 124) A1preferred YFW STCLIVM 1° Anchor A YFW PG G YFW 1° Anchor 10-mer (SEQ IDDEAS Y NO: 125) (SEQ ID NO: 126) deleterious RHK RHKDEPY P G PRHK QN FW(SEQ ID (SEO ID NO: 128) NO: 127) A2.1 preferred YFW 1° Anchor YFW STCYFW A P 1° Anchor 9-mer LMIVQAT VLIMAT deleterious DEP (SEQ ID DERKH RKHDERKH (SEQ ID NO: 130) NO: 129) (SEQ ID NO: 131) (SEQ ID NO: 132)Position: 1 2 3 4 5 6 7 8 9 C-Terminus A2.1 preferred AYFW 1° AnchorLVIM G G FYWL VIM 1° Anchor 10-mer LMIVQAT (SEQ ID (SEQ ID VLIMAT(SEQ ID NO: 133) NO: 134) NO: 135) (SEQ ID NO: 136) deleterious DEP DERKHA P RKH DERKH RKH (SEQ ID (SEQ ID NO: 137) NO: 138) A3 preferred RHK1° Anchor YFW PRHKYFW A YFW P 1° Anchor LMVISATFCGD (SEQ ID KYRHFA(SEQ ID NO: 139) NO: 140) (SEQ ID NO: 141) deleterious DEP DE A11preferred A 1° Anchor YFW YFW A YFW YFW P 1° Anchor VTLMISAGNCDF KRYH(SEQ ID NO: 142) (SEQ ID NO: 143) deleterious DEP A G A24 preferredYFWRHK 1° Anchor STC YFW YFW 1° Anchor 9-mer (SEQ ID YFWM FLIW NO: 144)(SEQ ID NO: 145) (SEQ ID NO: 146) deleterious DEG DE G QNP DERHK G AQN(SEQ ID NO: 147) A24 preferred 1° Anchor P YFWP P 1° Anchor 10-mer YFWM(SEQ ID FLIW (SEQ ID NO: 148) NO: 149) (SEQ ID NO: 150) deleterious GDEQN RHK DE A QN DEA A3101 preferred RHK 1° Anchor MVTALIS YFW P YFW YFWAP 1° Anchor RK deleterious DEP (SEQ ID NO: 151) DE ADE DE DE DE A3301preferred 1° Anchor YFW AYFW 1° Anchor MVALFIST (SEQ ID RK(SEQ ID NO: 152) NO: 153) deleterious GP DE A6801 preferred YFWSTC 1°Anchor YFWLIVM YFW P 1° Anchor (SEQ ID NO: 154) AVTMSL (SEQ ID RK(SEQ ID NO: 156) NO: 155) deleterious GP DEG RHK A B0702 preferredRHKFWY 1° Anchor RHK RHK RHK RHK PA 1° Anchor (SEQ ID NO: 157) PLMFWYAIV (SEQ ID NO: 158) deleterious DEQNP DEP DE DE GDE QN DE(SEQ ID NO: 159) B3501 preferred FWYLIVM 1° Anchor FWY FWY 1° Anchor(SEQ ID NO: 160) P LMFWYIVA (SEQ ID NO: 161) deleterious AGP G G B51preferred LIVMFWY 1° Anchor FWY STC FWY G FWY 1° Anchor (SEQ ID NO: 162)P LIVFWYAM (SEQ ID NO: 163) deleterious AGPDERHKSTC DE G DEQN GDE(SEQ ID NO: 164) (SEQ ID NO: 165) B5301 preferred LIVMFWY 1° Anchor FWYSTC FWY LIVMFWY FWY 1° Anchor (SEQ ID NO: 166) P (SEQ ID NO: 167)IMFWYALV (SEQ ID NO: 168) deleterious AGPQN G RHKQN DE (SEQ ID NO: 169)(SEQ ID NO: 170) B5401 preferred FWY 1° Anchor FWYLIVM LIVM ALIVM FWYAP1° Anchor P (SEQ ID (SEQ ID (SEQ ID NO: 173) (SEQ ID ATIVLMFWY NO: 171)NO: 172) NO: 174) (SEQ ID NO: 175) deleterious GPQNDE GDESTC RHKDE DEQNDGE DE (SEQ ID NO: 176) (SEQ ID (SEQ ID (SEQ ID NO: 179) NO: 177)NO: 178) Italicized residues indicate less preferred or “tolerated”residues The information in this Table is specific for 9-mers unlessotherwise specified.

TABLE V V1-A1-9mers: 161P5C5 Pos 123456789 Score 11 LIEVEFRDR 9.000Portion of SEQ ID NO: 4; each start 13 EVEFRDRQA 0.900position is specified, the length of each 58 DANMLAIYF 0.500peptide is 9 amino acids, the end 40 VVNDKPISF 0.500position for each peptide is the start 34 TNEFLTVVN 0.450position plus eight 8 LSMLIEVEF 0.300 41 VNDKPISFK 0.250 1 MLERGNVLS0.180 29 FSIIFTNEF 0.150 56 WFDANMLAI 0.125 20 QAYIRVRMF 0.100 61MLAIYFDHR 0.100 49 KACFNRQWF 0.100 46 ISFKACFNR 0.075 31 IIFTNEFLT 0.05057 FDANMLAIY 0.050 44 KPISFKACF 0.050 6 NVLSMLIEV 0.050 9 SMLIEVEFR0.050 16 FRDRQAYIR 0.050 33 FTNEFLTVV 0.025 38 LTVVNDKPI 0.025 25VRMFFSIIF 0.025 60 NMLAIYFDH 0.025 26 RMFFSIIFT 0.025 39 TVVNDKPIS 0.0204 RGNVLSMLI 0.013 30 SIIFTNEFL 0.010 62 LAIYFDHRM 0.010 63 AIYFDHRMH0.010 36 EFLTVVNDK 0.010 22 YIRVRMFFS 0.005 18 DRQAYIRVR 0.005 10MLIEVEFRD 0.005 19 RQAYIRVRM 0.003 14 VEFRDRQAY 0.003 42 NDKPISFKA 0.00321 AYIRVRMFF 0.003 32 IFTNEFLTV 0.003 27 MFFSIIFTN 0.003 55 QWFDANMLA0.003 5 GNVLSMLIE 0.001 45 PISFKACFN 0.001 37 FLTVVNDKP 0.001 53NRQWFDANM 0.001 7 VLSMLIEVE 0.001 50 ACFNRQWFD 0.001 24 RVRMFFSII 0.00112 IEVEFRDRQ 0.001 48 FKACFNRQW 0.001 3 ERGNVLSML 0.001 59 ANMLAIYFD0.001 43 DKPISFKAC 0.001 23 IRVRMFFSI 0.000 17 RDRQAYIRV 0.000 2LERGNVLSM 0.000 51 CFNRQWFDA 0.000 52 FNRQWFDAN 0.000 54 RQWFDANML 0.00015 EFRDRQAYI 0.000 35 NEFLTVVND 0.000 47 SFKACFNRQ 0.000 28 FFSIIFTNE0.000 V2-A1-9mers: 161P5C5 Pos 123456789 Score 5 KACFSRQWF 0.100Portion of SEQ ID NO: 6; each start 2 ISFKACFSR 0.075position is specified, the length of 8 FSRQWFDAN 0.002each peptide is 9 amino acids, the end 6 ACFSRQWFD 0.001position for each peptide is the start 9 SRQWFDANM 0.001position plus eight 1 PISFKACFS 0.001 4 FKACFSRQW 0.001 7 CFSRQWFDA0.000 3 SFKACFSRQ 0.000 V3-A1-9mers: 161P5C5 Pos 123456789 Score 4DANMLPIYF 0.500 Portion of SEQ ID NO: 8; each start 6 NMLPIYFDH 0.250position is specified, the length of each 2 WFDANMLPI 0.125peptide is 9 amino acids, the end 7 MLPIYFDHR 0.100position for each peptide is the start 3 FDANMLPIY 0.050position plus eight 8 LPIYFDHRM 0.003 9 PIYFDHRMH 0.001 5 ANMLPIYFD0.001 1 QWFDANMLP 0.000

TABLE VI V1-A1-10mers: 161P5C5 Pos 1234567890 Score 13 EVEFRDRQAY45.000  Portion of SEQ ID NO: 4; each start 56 WFDANMLAIY 2.500position is specified, the length of each 11 LIEVEFRDRQ 0.900peptide is 10 amino acids, the end 39 TVVNDKPISF 0.500position for each peptide is the start 20 QAYIRVRMFF 0.500position plus nine 1 MLERGNVLSM 0.450 7 VLSMLIEVEF 0.200 40 VVNDKPISFK0.200 8 LSMLIEVEFR 0.150 10 MLIEVEFRDR 0.100 41 VNDKPISFKA 0.062 38LTVVNDKPIS 0.050 31 IIFTNEFLTV 0.050 50 ACFNRQWFDA 0.050 60 NMLAIYFDHR0.050 45 PISFKACFNR 0.050 33 FTNEFLTVVN 0.050 24 RVRMFFSIIF 0.050 30SIIFTNEFLT 0.050 34 TNEFLTVVND 0.045 57 FDANMLAIYF 0.025 26 RMFFSIIFTN0.025 29 FSIIFTNEFL 0.015 19 RQAYIRVRMF 0.015 16 FRDRQAYIRV 0.013 59ANMLAIYFDH 0.013 5 GNVLSMLIEV 0.013 61 MLAIYFDHRM 0.010 43 DKPISFKACF0.010 35 NEFLTVVNDK 0.010 37 FLTVVNDKPI 0.010 62 LAIYFDHRMH 0.010 48FKACFNRQWF 0.005 28 FFSIIFTNEF 0.005 22 YIRVRMFFSI 0.005 9 SMLIEVEFRD0.003 55 QWFDANMLAI 0.003 44 KPISFKACFN 0.003 3 ERGNVLSMLI 0.003 25VRMFFSIIFT 0.003 58 DANMLAIYFD 0.002 46 ISFKACFNRQ 0.002 4 RGNVLSMLIE0.001 15 EFRDRQAYIR 0.001 6 NVLSMLIEVE 0.001 18 DRQAYIRVRM 0.001 49KACFNRQWFD 0.001 54 RQWFDANMLA 0.001 42 NDKPISFKAC 0.001 17 RDRQAYIRVR0.001 52 FNRQWFDANM 0.001 32 IFTNEFLTVV 0.001 53 NRQWFDANML 0.001 12IEVEFRDRQA 0.001 21 AYIRVRMFFS 0.000 14 VEFRDRQAYI 0.000 47 SFKACFNRQW0.000 2 LERGNVLSML 0.000 27 MFFSIIFTNE 0.000 23 IRVRMFFSII 0.000 51CFNRQWFDAN 0.000 36 EFLTVVNDKP 0.000 V2-A1-10mers: 161P5C5 Pos1234567890 Score 2 PISFKACFSR 0.050 Portion of SEQ ID NO: 6; each start7 ACFSRQWFDA 0.050 position is specified, the length of each 5FKACFSRQWF 0.005 peptide is 10 amino acids, the end 9 FSRQWFDANM 0.003position for each peptide is the start 1 KPISFKACFS 0.003position plus nine 3 ISFKACFSRQ 0.002 6 KACFSRQWFD 0.001 10 SRQWFDANML0.001 8 CFSRQWFDAN 0.000 4 SFKACFSRQW 0.000 V3-A1-10mers: 161P5C5 Pos1234567890 Score 3 WFDANMLPIY 2.500 Portion of SEQ ID NO: 8; each start7 NMLPIYFDHR 0.500 position is specified, the length of each 4FDANMLPIYF 0.025 peptide is 10 amino acids, the end 6 ANMLPIYFDH 0.013position for each peptide is the start 8 MLPIYFDHRM 0.010position plus nine 2 QWFDANMLPI 0.003 9 LPIYFDHRMH 0.003 5 DANMLPIYFD0.002 1 RQWFDANMLP 0.000

TABLE VII V1-A2-9mers: 161P5C5 Pos 123456789 Score 26 RMFFSIIFT 251.905Portion 33 FTNEFLTVV 65.285 of SEQ 6 NVLSMLIEV 51.790 ID 31 IIFTNEFLT37.381 NO: 4; 54 RQWFDANML 17.977 each 30 SIIFTNEFL 7.916 start 60NMLAIYFDH 1.732 position 10 MLIEVEFRD 0.603 is 19 RQAYIRVRM 0.504specified, 62 LAIYFDHRM 0.446 the 22 YIRVRMFFS 0.263 length 9 SMLIEVEFR0.247 of each 38 LTVVNDKPI 0.246 peptide 57 FDANMLAIY 0.000 is 9 58DANMLAIYF 0.000 amino 13 EVEFRDRQA 0.000 acids, 48 FKACFNRQW 0.000 theend 52 FNRQWFDAN 0.000 position 25 VRMFFSIIF 0.000 for 5 GNVLSMLIE 0.000each 28 FFSIIFTNE 0.000 peptide 11 LIEVEFRDR 0.000 is the 21 AYIRVRMFF0.000 start 34 TNEFLTVVN 0.000 position 36 EFLTVVNDK 0.000 plus 16FRDRQAYIR 0.000 eight 47 SFKACFNRQ 0.000 18 DRQAYIRVR 0.000 V2-A2-9mers:161P5C5 Pos 123456789 Score 7 CFSRQWFDA 0.034 Portion of 5 KACFSRQWF0.020 SEQ ID NO: 6; 6 ACFSRQWFD 0.015 each start position 2 ISFKACFSR0.004 is specified, the 9 SRQWFDANM 0.003 length of each peptide 1PISFKACFS 0.001 is 9 amino acids, the end 4 FKACFSRQW 0.000 position foreach 8 FSRQWFDAN 0.000 peptide is the 3 SFKACFSRQ 0.000 start positionplus eight V3-A2-9mers: 161P5C5 Pos 123456789 Score 6 NMLPIYFDH 1.732Portion of 8 LPIYFDHRM 0.209 SEQ ID NO: 8; 2 WFDANMLPI 0.031 each startposition 7 MLPIYFDHR 0.024 is specified, 5 ANMLPIYFD 0.005 the length ofeach 3 FDANMLPIY 0.000 peptide is 9 amino 4 DANMLPIYF 0.000 acids, theend 9 PIYFDHRMH 0.000 position for each 1 QWFDANMLP 0.000 peptide is thestart position plus eight

TABLE VIII V1-A2-10mers: 161P5C5 Pos 1234567890 Score 31 IIFTNEFLTV114.292 Portion of 37 FLTVVNDKPI 47.991 SEQ ID NO: 4; 61 MLAIYFDHRM32.093 each start 14 VEFRDRQAYI 7.018 position 30 SIIFTNEFLT 5.943 is 22YIRVRMFFSI 5.527 specified, 54 RQWFDANMLA 4.181 the length 26 RMFFSIIFTN2.656 of each 29 FSIIFTNEFL 1.729 peptide is 1 MLERGNVLSM 1.243 10 amino50 ACFNRQWFDA 1.183 acids, the 5 GNVLSMLIEV 1.044 end 32 IFTNEFLTVV0.294 position 27 MFFSIIFTNE 0.000 for each 3 ERGNVLSMLI 0.000 peptideis 4 RGNVLSMLIE 0.000 the start 56 WFDANMLAIY 0.000 position 18DRQAYIRVRM 0.000 Plus nine 51 CFNRQWFDAN 0.000 13 EVEFRDRQAY 0.000 43DKPISFKACF 0.000 34 TNEFLTVVND 0.000 36 EFLTVVNDKP 0.000 47 SFKACFNRQW0.000 17 RDRQAYIRVR 0.000 15 EFRDRQAYIR 0.000 V2-A2-10mers: 161P5C5 Pos1234567890 Score 7 ACFSRQWFDA 1.183 Portion of 9 FSRQWFDANM 0.043 SEQ IDNO: 6; 6 KACFSRQWFD 0.030 each start position 1 KPISFKACFS 0.009 isspecified, the length 10 SRQWFDANML 0.003 of each peptide is 5FKACFSRQWF 0.003 10 amino acids, 3 ISFKACFSRQ 0.001 the end position 2PISFKACFSR 0.000 for each peptide is the 8 CFSRQWFDAN 0.000 startposition plus nine 4 SFKACFSRQW 0.000 V3-A2-10mers: 161P5C5 Pos1234567890 Score 8 MLPIYFDHRM 32.093 Portion of 2 QWFDANMLPI 0.051 SEQID NO: 8; 7 NMLPIYFDHR 0.037 each start position 6 ANMLPIYFDH 0.016 isspecified, the length 1 RQWFDANMLP 0.013 of each peptide is 4 FDANMLPIYF0.003 10 amino acids, 5 DANMLPIYFD 0.000 the end position for 9LPIYFDHRMH 0.000 each peptide is the 3 WFDANMLPIY 0.000 start positionplus nine

TABLE IX V1-A3-9mers: 161P5C5 Pos 123456789 Score 61 MLAIYFDHR 36.000Portion of 9 SMLIEVEFR 18.000 SEQ ID 26 RMFFSIIFT 1.500 NO: 4; each 60NMLAIYFDH 1.350 start 24 RVRMFFSII 0.540 position 40 VVNDKPISF 0.400 is46 ISFKACFNR 0.300 specified, 44 KPISFKACF 0.270 the length 10 MLIEVEFRD0.203 of each 31 IIFTNEFLT 0.150 peptide is 11 LIEVEFRDR 0.120 9 amino41 VNDKPISFK 0.090 acids, the 6 NVLSMLIEV 0.090 end 30 SIIFTNEFL 0.090position 54 RQWFDANML 0.090 for each 33 FTNEFLTVV 0.068 peptide is 49KACFNRQWF 0.060 the start 14 VEFRDRQAY 0.060 position 29 FSIIFTNEF 0.045plus eight 36 EFLTVVNDK 0.041 1 MLERGNVLS 0.040 20 QAYIRVRMF 0.030 7VLSMLIEVE 0.030 37 FLTVVNDKP 0.030 8 LSMLIEVEF 0.022 38 LTVVNDKPI 0.02225 VRMFFSIIF 0.018 22 YIRVRMFFS 0.018 16 FRDRQAYIR 0.012 58 DANMLAIYF0.012 63 AIYFDHRMH 0.010 21 AYIRVRMFF 0.009 62 LAIYFDHRM 0.009 19RQAYIRVRM 0.009 23 IRVRMFFSI 0.008 57 FDANMLAIY 0.006 39 TVVNDKPIS 0.0062 LERGNVLSM 0.005 50 ACFNRQWFD 0.003 55 QWFDANMLA 0.002 56 WFDANMLAI0.002 42 NDKPISFKA 0.001 32 IFTNEFLTV 0.001 4 RGNVLSMLI 0.001 35NEFLTVVND 0.001 27 MFFSIIFTN 0.001 13 EVEFRDRQA 0.001 51 CFNRQWFDA 0.00153 NRQWFDANM 0.001 18 DRQAYIRVR 0.001 5 GNVLSMLIE 0.001 12 IEVEFRDRQ0.000 3 ERGNVLSML 0.000 17 RDRQAYIRV 0.000 52 FNRQWFDAN 0.000 59ANMLAIYFD 0.000 45 PISFKACFN 0.000 15 EFRDRQAYI 0.000 28 FFSIIFTNE 0.00043 DKPISFKAC 0.000 48 FKACFNRQW 0.000 47 SFKACFNRQ 0.000 34 TNEFLTVVN0.000 V2-A3-9mers: 161P5C5 Pos 123456789 Score 2 ISFKACFSR 0.300 Portionof 5 KACFSRQWF 0.060 SEQ ID NO: 6; 6 ACFSRQWFD 0.003 each start position8 FSRQWFDAN 0.001 is specified, 7 CFSRQWFDA 0.001 the length of each 9SRQWFDANM 0.001 peptide is 9 amino acids, 1 PISFKACFS 0.000 the endposition for 3 SFKACFSRQ 0.000 each peptide is the 4 FKACFSRQW 0.000start position plus eight V3-A3-9mers: 161P5C5 Pos 123456789 Score 7MLPIYFDHR 36.000 Portion of 6 NMLPIYFDH 2.025 SEQ ID NO: 8; 4 DANMLPIYF0.012 each start position 8 LPIYFDHRM 0.009 is specified, 3 FDANMLPIY0.006 the length of each 2 WFDANMLPI 0.002 peptide is 9 amino 9PIYFDHRMH 0.001 acids, the end 5 ANMLPIYFD 0.000 position for each 1QWFDANMLP 0.000 peptide is the start position plus eight

TABLE X V1-A3-10mers: 161P5C5 Pos 1234567890 Score 60 NMLAIYFDHR 54.000Portion 40 VVNDKPISFK 4.500 of SEQ 7 VLSMLIEVEF 3.000 ID 10 MLIEVEFRDR2.700 NO: 4; 35 NEFLTVVNDK 1.350 each 1 MLERGNVLSM 1.200 start 24RVRMFFSIIF 1.200 position 26 RMFFSIIFTN 0.675 is 31 IIFTNEFLTV 0.600specified, 61 MLAIYFDHRM 0.600 the 39 TVVNDKPISF 0.600 length 22YIRVRMFFSI 0.540 of each 37 FLTVVNDKPI 0.300 peptide 20 QAYIRVRMFF 0.300is 10 9 SMLIEVEFRD 0.135 amino 13 EVEFRDRQAY 0.120 acids, 45 PISFKACFNR0.120 the end 8 LSMLIEVEFR 0.090 position 54 RQWFDANMLA 0.060 for 30SIIFTNEFLT 0.045 each 50 ACFNRQWFDA 0.030 peptide 19 RQAYIRVRMF 0.018 isthe 14 VEFRDRQAYI 0.009 start 55 QWFDANMLAI 0.009 position 23 IRVRMFFSII0.008 plus 2 LERGNVLSML 0.006 nine 28 FFSIIFTNEF 0.006 56 WFDANMLAIY0.006 11 LIEVEFRDRQ 0.006 5 GNVLSMLIEV 0.005 6 NVLSMLIEVE 0.005 29FSIIFTNEFL 0.005 57 FDANMLAIYF 0.004 15 EFRDRQAYIR 0.004 38 LTVVNDKPIS0.003 59 ANMLAIYFDH 0.003 48 FKACFNRQWF 0.002 17 RDRQAYIRVR 0.002 41VNDKPISFKA 0.002 49 KACFNRQWFD 0.002 43 DKPISFKACF 0.002 33 FTNEFLTVVN0.002 46 ISFKACFNRQ 0.002 52 FNRQWFDANM 0.001 32 IFTNEFLTVV 0.001 44KPISFKACFN 0.001 53 NRQWFDANML 0.001 25 VRMFFSIIFT 0.000 42 NDKPISFKAC0.000 27 MFFSIIFTNE 0.000 16 FRDRQAYIRV 0.000 3 ERGNVLSMLI 0.000 62LAIYFDHRMH 0.000 21 AYIRVRMFFS 0.000 58 DANMLAIYFD 0.000 51 CFNRQWFDAN0.000 12 IEVEFRDRQA 0.000 34 TNEFLTVVND 0.000 18 DRQAYIRVRM 0.000 47SFKACFNRQW 0.000 4 RGNVLSMLIE 0.000 36 EFLTVVNDKP 0.000 V2-A3-10mers:161P5C5 Pos 1234567890 Score 2 PISFKACFSR 0.120 Portion of 7 ACFSRQWFDA0.030 SEQ ID NO: 6; 9 FSRQWFDANM 0.003 each start position 5 FKACFSRQWF0.002 is specified, the length 6 KACFSRQWFD 0.002 of each peptide is 1KPISFKACFS 0.002 10 amino acids, the end 3 ISFKACFSRQ 0.002 position foreach peptide 10 SRQWFDANML 0.001 is the start position 8 CFSRQWFDAN0.000 plus nine 4 SFKACFSRQW 0.000 V3-A3-10mers: 161P5C5 Pos 1234567890Score 7 NMLPIYFDHR 81.000 Portion of 8 MLPIYFDHRM 0.600 SEQ ID NO: 8; 2QWFDANMLPI 0.009 each start position 3 WFDANMLPIY 0.006 is specified,the length 1 RQWFDANMLP 0.006 of each peptide is 4 FDANMLPIYF 0.004 10amino acids, the end 6 ANMLPIYFDH 0.003 position for each peptide 5DANMLPIYFD 0.000 is the start position 9 LPIYFDHRMH 0.000 plus nine

TABLE XI V1-A11-9mers: 161P5C5 Pos 123456789 Score 9 SMLIEVEFR 0.120Portion 36 EFLTVVNDK 0.090 of SEQ 61 MLAIYFDHR 0.080 ID 24 RVRMFFSII0.060 NO: 4; 6 NVLSMLIEV 0.060 each 41 VNDKPISFK 0.040 start 40VVNDKPISF 0.040 position 54 RQWFDANML 0.036 is 46 ISFKACFNR 0.024specified, 19 RQAYIRVRM 0.018 the 60 NMLAIYFDH 0.018 length 21 AYIRVRMFF0.012 of each 33 FTNEFLTVV 0.010 peptide 44 KPISFKACF 0.009 is 9 11LIEVEFRDR 0.008 amino 16 FRDRQAYIR 0.008 acids, 38 LTVVNDKPI 0.007 theend 51 CFNRQWFDA 0.006 position 30 SIIFTNEFL 0.006 for 49 KACFNRQWF0.006 each 26 RMFFSIIFT 0.005 peptide 32 IFTNEFLTV 0.004 is the 56WFDANMLAI 0.004 start 39 TVVNDKPIS 0.003 position 62 LAIYFDHRM 0.003plus 31 IIFTNEFLT 0.002 eight 10 MLIEVEFRD 0.002 22 YIRVRMFFS 0.001 4RGNVLSMLI 0.001 27 MFFSIIFTN 0.001 17 RDRQAYIRV 0.001 58 DANMLAIYF 0.00114 VEFRDRQAY 0.001 2 LERGNVLSM 0.001 23 IRVRMFFSI 0.001 25 VRMFFSIIF0.001 55 QWFDANMLA 0.001 63 AIYFDHRMH 0.001 50 ACFNRQWFD 0.001 42NDKPISFKA 0.001 13 EVEFRDRQA 0.001 15 EFRDRQAYI 0.001 1 MLERGNVLS 0.0008 LSMLIEVEF 0.000 37 FLTVVNDKP 0.000 7 VLSMLIEVE 0.000 20 QAYIRVRMF0.000 5 GNVLSMLIE 0.000 29 FSIIFTNEF 0.000 57 FDANMLAIY 0.000 47SFKACFNRQ 0.000 53 NRQWFDANM 0.000 28 FFSIIFTNE 0.000 59 ANMLAIYFD 0.00035 NEFLTVVND 0.000 18 DRQAYIRVR 0.000 12 IEVEFRDRQ 0.000 3 ERGNVLSML0.000 52 FNRQWFDAN 0.000 45 PISFKACFN 0.000 34 TNEFLTVVN 0.000 48FKACFNRQW 0.000 43 DKPISFKAC 0.000 V2-A11-9mers: 161P5C5 Pos 123456789Score 2 ISFKACFSR 0.024 Portion of 5 KACFSRQWF 0.006 SEQ ID NO: 6; 7CFSRQWFDA 0.006 each start position 6 ACFSRQWFD 0.001 is specified, thelength 9 SRQWFDANM 0.000 of each peptide is 3 SFKACFSRQ 0.000 9 aminoacids, the end 1 PISFKACFS 0.000 position for each 4 FKACFSRQW 0.000peptide is the start 8 FSRQWFDAN 0.000 position plus eight V3-A11-9mers:161P5C5 Pos 123456789 Score 7 MLPIYFDHR 0.080 Portion of 6 NMLPIYFDH0.018 SEQ ID NO: 8; 2 WFDANMLPI 0.004 each start position 8 LPIYFDHRM0.003 is specified, the length 4 DANMLPIYF 0.001 of each peptide is 3FDANMLPIY 0.000 9 amino acids, the end 5 ANMLPIYFD 0.000 position foreach 9 PIYFDHRMH 0.000 peptide is the start 1 QWFDANMLP 0.000 positionplus eight

TABLE XII V1-A11-10mers: 161P5C5 Pos 1234567890 Score 40 VVNDKPISFK2.000 Portion 60 NMLAIYFDHR 0.120 of SEQ 24 RVRMFFSIIF 0.120 ID 35NEFLTVVNDK 0.120 NO: 4; 54 RQWFDANMLA 0.072 each 39 TVVNDKPISF 0.060start 45 PISFKACFNR 0.024 position 15 EFRDRQAYIR 0.024 is 31 IIFTNEFLTV0.016 specified, 10 MLIEVEFRDR 0.012 the 22 YIRVRMFFSI 0.012 length 50ACFNRQWFDA 0.012 of each 20 QAYIRVRMFF 0.008 peptide 8 LSMLIEVEFR 0.008is 10 1 MLERGNVLSM 0.008 amino 26 RMFFSIIFTN 0.007 acids, 13 EVEFRDRQAY0.006 the end 61 MLAIYFDHRM 0.004 position 7 VLSMLIEVEF 0.004 for 5GNVLSMLIEV 0.004 each 6 NVLSMLIEVE 0.003 peptide 59 ANMLAIYFDH 0.002 isthe 37 FLTVVNDKPI 0.002 start 32 IFTNEFLTVV 0.002 position 56 WFDANMLAIY0.002 plus 28 FESIIFTNEF 0.002 nine 21 AYIRVRMFFS 0.002 9 SMLIEVEFRD0.002 19 RQAYIRVRMF 0.002 30 SIIFTNEFLT 0.002 38 LTVVNDKPIS 0.002 41VNDKPISFKA 0.001 17 RDRQAYIRVR 0.001 49 KACFNRQWFD 0.001 14 VEFRDRQAYI0.001 33 FTNEFLTVVN 0.001 44 KPISFKACFN 0.001 55 QWFDANMLAI 0.001 2LERGNVLSML 0.001 57 FDANMLAIYF 0.000 52 FNRQWFDANM 0.000 11 LIEVEFRDRQ0.000 27 MFFSIIFTNE 0.000 16 FRDRQAYIRV 0.000 62 LAIYFDHRMH 0.000 23IRVRMFFSII 0.000 29 FSIIFTNEFL 0.000 51 CFNRQWFDAN 0.000 48 FKACFNRQWF0.000 47 SFKACFNRQW 0.000 53 NRQWFDANML 0.000 4 RGNVLSMLIE 0.000 58DANMLAIYFD 0.000 3 ERGNVLSMLI 0.000 36 EFLTVVNDKP 0.000 12 IEVEFRDRQA0.000 25 VRMFFSIIFT 0.000 18 DRQAYIRVRM 0.000 43 DKPISFKACF 0.000 46ISFKACFNRQ 0.000 34 TNEFLTVVND 0.000 42 NDKPISFKAC 0.000 V2-A11-10mers:161P5C5 Pos 1234567890 Score 2 PISFKACFSR 0.024 Portion of 7 ACFSRQWFDA0.012 SEQ ID NO: 6; 6 KACFSRQWFD 0.001 each start position 1 KPISFKACFS0.001 is specified, the length 8 CFSRQWFDAN 0.000 of each peptide is 10SRQWFDANML 0.000 10 amino acids, the end 4 SFKACFSRQW 0.000 position foreach 9 FSRQWFDANM 0.000 peptide is the start 5 FKACFSRQWF 0.000 positionplus nine 3 ISFKACFSRQ 0.000 V3-A11-10mers: 161P5C5 Pos 1234567890 Score7 NMLPIYFDHR 0.120 Portion of 1 RQWFDANMLP 0.007 SEQ ID NO: 8; 8MLPIYFDHRM 0.004 each start position 6 ANMLPIYFDH 0.002 is specified,the length 3 WFDANMLPIY 0.002 of each peptide is 2 QWFDANMLPI 0.001 10amino acids, the end 4 FDANMLPIYF 0.000 position for each 9 LPIYFDHRMH0.000 peptide is the start 5 DANMLPIYFD 0.000 position plus nine

TABLE XIII V1-A24-9mers Pos 123456789 Score 21 AYIRVRMFF 210.000 Portion54 RQWFDANML 9.600 of SEQ 30 SIIFTNEFL 6.000 ID 44 KPISFKACF 6.000 NO:4; 15 EFRDRQAYI 6.000 each 56 WFDANMLAI 5.000 start 49 KACFNRQWF 4.800position 8 LSMLIEVEF 4.620 is 29 FSIIFTNEF 3.960 specified, 40 VVNDKPISF3.600 the 58 DANMLAIYF 3.600 length 4 RGNVLSMLI 3.600 of each 24RVRMFFSII 2.400 peptide 20 QAYIRVRMF 2.000 is 9 38 LTVVNDKPI 1.500 amino19 RQAYIRVRM 1.400 acids, 51 CFNRQWFDA 0.750 the end 62 LAIYFDHRM 0.750position 27 MFFSIIFTN 0.700 for 32 IFTNEFLTV 0.600 each 3 ERGNVLSML0.560 peptide 25 VRMFFSIIF 0.300 is the 26 RMFFSIIFT 0.200 start 23IRVRMFFSI 0.180 position 33 FTNEFLTVV 0.180 plus 34 TNEFLTVVN 0.180eight 6 NVLSMLIEV 0.165 1 MLERGNVLS 0.150 13 EVEFRDRQA 0.150 39TVVNDKPIS 0.150 36 EFLTVVNDK 0.126 52 FNRQWFDAN 0.120 55 QWFDANMLA 0.12031 IIFTNEFLT 0.100 22 YIRVRMFFS 0.100 28 FFSIIFTNE 0.084 53 NRQWFDANM0.075 47 SFKACFNRQ 0.072 2 LERGNVLSM 0.050 10 MLIEVEFRD 0.025 60NMLAIYFDH 0.021 17 RDRQAYIRV 0.020 42 NDKPISFKA 0.018 43 DKPISFKAC 0.01811 LIEVEFRDR 0.018 37 FLTVVNDKP 0.015 59 ANMLAIYFD 0.015 5 GNVLSMLIE0.015 9 SMLIEVEFR 0.015 61 MLAIYFDHR 0.012 7 VLSMLIEVE 0.012 14VEFRDRQAY 0.012 63 AIYFDHRMH 0.010 48 FKACFNRQW 0.010 45 PISFKACFN 0.01050 ACFNRQWFD 0.010 41 VNDKPISFK 0.010 57 FDANMLAIY 0.010 46 ISFKACFNR0.010 12 IEVEFRDRQ 0.002 18 DRQAYIRVR 0.002 16 FRDRQAYIR 0.001 35NEFLTVVND 0.001 V2-A24-9mers: 161P5C5 Pos 123456789 Score 5 KACFSRQWF4.800 Portion of 7 CFSRQWFDA 0.500 SEQ ID NO: 6; 8 FSRQWFDAN 0.120 eachstart position 9 SRQWFDANM 0.075 is specified, the length 3 SFKACFSRQ0.060 of each peptide is 4 FKACFSRQW 0.010 9 amino acids, 6 ACFSRQWFD0.010 the end position 2 ISFKACFSR 0.010 for each peptide 1 PISFKACFS0.010 is the start position plus eight V3-A24-9mers: 161P5C5 Pos123456789 Score 2 WFDANMLPI 5.000 Portion of 4 DANMLPIYF 3.600 SEQ IDNO: 8; 8 LPIYFDHRM 0.750 each start position 6 NMLPIYFDH 0.025 isspecified, the length 7 MLPIYFDHR 0.018 of each peptide is 5 ANMLPIYFD0.015 9 amino acids, 1 QWFDANMLP 0.012 the end position 3 FDANMLPIY0.010 for each peptide 9 PIYFDHRMH 0.001 is the start position pluseight

TABLE XIV V1-A24-10mers: 161P5C5 Pos 1234567890 Score 28 FFSIIFTNEF13.200 Portion 21 AYIRVRMFFS 7.500 of SEQ ID 29 FSIIFTNEFL 6.000 NO: 4;19 RQAYIRVRMF 4.000 each 24 RVRMFFSIIF 4.000 start 7 VLSMLIEVEF 3.080position 39 TVVNDKPISF 3.000 is 20 QAYIRVRMFF 2.800 specified, 55QWFDANMLAI 1.200 the 22 YIRVRMFFSI 1.200 length of 37 FLTVVNDKPI 1.000each 51 CFNRQWFDAN 0.900 peptide 1 MLERGNVLSM 0.750 is 10 53 NRQWFDANML0.720 amino 2 LERGNVLSML 0.560 acids, 32 IFTNEFLTVV 0.500 the end 56WFDANMLAIY 0.500 position 61 MLAIYFDHRM 0.500 for each 47 SFKACFNRQW0.500 peptide 52 FNRQWFDANM 0.500 is the 44 KPISFKACFN 0.300 start 43DKPISFKACF 0.300 position 26 RMFFSIIFTN 0.280 plus nine 48 FKACFNRQWF0.240 57 FDANMLAIYF 0.240 33 FTNEFLTVVN 0.216 54 RQWFDANMLA 0.200 23IRVRMFFSII 0.180 13 EVEFRDRQAY 0.180 5 GNVLSMLIEV 0.165 41 VNDKPISFKA0.154 38 LTVVNDKPIS 0.150 30 SIIFTNEFLT 0.150 3 ERGNVLSMLI 0.120 31IIFTNEFLTV 0.120 36 EFLTVVNDKP 0.116 18 DRQAYIRVRM 0.105 50 ACFNRQWFDA0.100 14 VEFRDRQAYI 0.100 27 MFFSIIFTNE 0.084 15 EFRDRQAYIR 0.060 4RGNVLSMLIE 0.030 10 MLIEVEFRDR 0.022 59 ANMLAIYFDH 0.021 9 SMLIEVEFRD0.021 49 KACFNRQWFD 0.020 12 IEVEFRDRQA 0.018 40 VVNDKPISFK 0.018 6NVLSMLIEVE 0.018 60 NMLAIYFDHR 0.018 62 LAIYFDHRMH 0.015 8 LSMLIEVEFR0.015 11 LIEVEFRDRQ 0.015 34 TNEFLTVVND 0.015 58 DANMLAIYFD 0.015 25VRMFFSIIFT 0.015 42 NDKPISFKAC 0.014 46 ISFKACFNRQ 0.014 16 FRDRQAYIRV0.010 17 RDRQAYIRVR 0.002 35 NEFLTVVNDK 0.002 45 PISFKACFNR 0.001V2-A24-10mers: 161P5C5 Pos 1234567890 Score 10 SRQWFDANML 0.720 Portionof 8 CFSRQWFDAN 0.600 SEQ ID NO: 6; 4 SFKACFSRQW 0.500 each startposition is 9 FSRQWFDANM 0.500 specified, the length 1 KPISFKACFS 0.300of each peptide is 5 FKACFSRQWF 0.240 10 amino acids, 7 ACFSRQWFDA 0.100the end position 6 KACFSRQWFD 0.020 for each peptide 3 ISFKACFSRQ 0.012is the start position 2 PISFKACFSR 0.001 plus nine V3-A24-10mers:161P5C5 Pos 1234567890 Score 2 QWFDANMLPI 1.200 Portion of 8 MLPIYFDHRM0.750 SEQ ID NO: 8; 3 WFDANMLPIY 0.500 each start position is 4FDANMLPIYF 0.240 specified, the length 7 NMLPIYFDHR 0.022 of eachpeptide is 6 ANMLPIYFDH 0.021 10 amino acids, 1 RQWFDANMLP 0.020 the endposition 9 LPIYFDHRMH 0.015 for each peptide 5 DANMLPIYFD 0.015 is thestart position plus nine

TABLE XV V1-B7-9mers: 161P5C5 Pos 123456789 Score 24 RVRMFFSII 20.000Portion of 54 RQWFDANML 4.000 SEQ ID NO: 4; 30 SIIFTNEFL 4.000 eachstart position 62 LAIYFDHRM 3.000 is specified, the length 19 RQAYIRVRM1.500 of each peptide is 6 NVLSMLIEV 1.000 9 amino acids, the end 2LERGNVLSM 1.000 position for each 3 ERGNVLSML 0.400 peptide is the start38 LTVVNDKPI 0.400 position plus eight 15 EFRDRQAYI 0.400 4 RGNVLSMLI0.400 44 KPISFKACF 0.400 22 YIRVRMFFS 0.200 17 RDRQAYIRV 0.200 33FTNEFLTVV 0.200 52 FNRQWFDAN 0.200 13 EVEFRDRQA 0.150 26 RMFFSIIFT 0.10039 TVVNDKPIS 0.100 31 IIFTNEFLT 0.100 53 NRQWFDANM 0.100 40 VVNDKPISF0.100 49 KACFNRQWF 0.090 59 ANMLAIYFD 0.090 20 QAYIRVRMF 0.060 8LSMLIEVEF 0.060 58 DANMLAIYF 0.060 23 IRVRMFFSI 0.040 63 AIYFDHRMH 0.03050 ACFNRQWFD 0.030 29 FSIIFTNEF 0.020 32 IFTNEFLTV 0.020 56 WFDANMLAI0.012 42 NDKPISFKA 0.010 61 MLAIYFDHR 0.010 9 SMLIEVEFR 0.010 5GNVLSMLIE 0.010 43 DKPISFKAC 0.010 46 ISFKACFNR 0.010 7 VLSMLIEVE 0.01055 QWFDANMLA 0.010 10 MLIEVEFRD 0.010 37 FLTVVNDKP 0.010 60 NMLAIYFDH0.010 51 CFNRQWFDA 0.010 21 AYIRVRMFF 0.009 25 VRMFFSIIF 0.006 1MLERGNVLS 0.006 34 TNEFLTVVN 0.006 41 VNDKPISFK 0.004 V2-B7-9mers Pos123456789 Score 8 FSRQWFDAN 0.200 Portion of 9 SRQWFDANM 0.100 SEQ IDNO: 6; 5 KACFSRQWF 0.090 each start position 6 ACFSRQWFD 0.030 isspecified, the length 7 CFSRQWFDA 0.010 of each peptide is 2 ISFKACFSR0.010 9 amino acids, the end 4 FKACFSRQW 0.002 position for each 1PISFKACFS 0.002 peptide is the start 3 SFKACFSRQ 0.001 position pluseight V3-B7-9mers: 161P5C5 Pos 123456789 Score 8 LPIYFDHRM 20.000Portion of 5 ANMLPIYFD 0.135 SEQ ID NO: 8; 4 DANMLPIYF 0.060 each startposition 2 WFDANMLPI 0.012 is specified, the length 7 MLPIYFDHR 0.010 ofeach peptide is 6 NMLPIYFDH 0.010 9 amino acids, the end 3 FDANMLPIY0.002 position for each 9 PIYFDHRMH 0.001 peptide is the start 1QWFDANMLP 0.001 position plus eight

TABLE XVI V1-B7-10mers: 161P5C5 Pos 1234567890 Score 52 FNRQWFDANM10.000 Portion 2 LERGNVLSML 4.000 of SEQ 22 YIRVRMFFSI 4.000 ID 29FSIIFTNEFL 4.000 NO: 4; 61 MLAIYFDHRM 1.000 each 24 RVRMFFSIIF 1.000start 53 NRQWFDANML 0.400 position 50 ACFNRQWFDA 0.300 is 1 MLERGNVLSM0.300 specified, 31 IIFTNEFLTV 0.200 the 5 GNVLSMLIEV 0.200 length 18DRQAYIRVRM 0.150 of each 30 SIIFTNEFLT 0.100 peptide 39 TVVNDKPISF 0.100is 10 54 RQWFDANMLA 0.100 amino 59 ANMLAIYFDH 0.090 acids, 20 QAYIRVRMFF0.090 the end 40 VVNDKPISFK 0.075 position 6 NVLSMLIEVE 0.050 for 13EVEFRDRQAY 0.045 each 55 QWFDANMLAI 0.040 peptide 14 VEFRDRQAYI 0.040 isthe 3 ERGNVLSMLI 0.040 start 23 IRVRMFFSII 0.040 position 25 VRMFFSIIFT0.030 plus 8 LSMLIEVEFR 0.030 nine 49 KACFNRQWFD 0.030 58 DANMLAIYFD0.030 62 LAIYFDHRMH 0.030 41 VNDKPISFKA 0.030 7 VLSMLIEVEF 0.020 26RMFFSIIFTN 0.020 19 RQAYIRVRMF 0.020 33 FTNEFLTVVN 0.020 38 LTVVNDKPIS0.020 32 IFTNEFLTVV 0.020 9 SMLIEVEFRD 0.010 12 IEVEFRDRQA 0.010 46ISFKACFNRQ 0.010 42 NDKPISFKAC 0.010 60 NMLAIYFDHR 0.010 17 RDRQAYIRVR0.010 10 MLIEVEFRDR 0.010 4 RGNVLSMLIE 0.010 15 EFRDRQAYIR 0.010 21AYIRVRMFFS 0.006 16 FRDRQAYIRV 0.006 11 LIEVEFRDRQ 0.004 48 FKACFNRQWF0.003 34 TNEFLTVVND 0.003 57 FDANMLAIYF 0.002 43 DKPISFKACF 0.002 51CFNRQWFDAN 0.002 47 SFKACFNRQW 0.002 28 FFSIIFTNEF 0.002 36 EFLTVVNDKP0.001 27 MFFSIIFTNE 0.001 35 NEFLTVVNDK 0.001 45 PISFKACFNR 0.001 56WFDANMLAIY 0.001 V2-B7-10mers: 161P5C5 Pos 1234567890 Score 9 FSRQWFDANM10.000 Portion of 1 KPISFKACFS 0.400 SEQ ID NO: 6; 10 SRQWFDANML 0.400each start position 7 ACFSRQWFDA 0.300 is specified, the length 6KACFSRQWFD 0.030 of each peptide is 3 ISFKACFSRQ 0.010 10 amino acids,the end 5 FKACFSRQWF 0.003 position for each 8 CFSRQWFDAN 0.002 peptideis the start 4 SFKACFSRQW 0.002 position plus nine 2 PISFKACFSR 0.001V3-B7-10mers: 161P5C5 Pos 1234567890 Score 8 MLPIYFDHRM 1.000 Portion of9 LPIYFDHRMH 0.200 SEQ ID NO: 8; 6 ANMLPIYFDH 0.090 each start position5 DANMLPIYFD 0.045 is specified, the length 2 QWFDANMLPI 0.040 of eachpeptide is 7 NMLPIYFDHR 0.010 10 amino acids, the end 1 RQWFDANMLP 0.010position for each 4 FDANMLPIYF 0.002 peptide is the start 3 WFDANMLPIY0.001 position plus nine

TABLE XVII V1-B35-9mers: 161P5C5 Pos 123456789 Score 44 KPISFKACF 40.000Portion 62 LAIYFDHRM 6.000 of SEQ 49 KACFNRQWF 6.000 ID NO: 4; 29FSIIFTNEF 5.000 each 8 LSMLIEVEF 5.000 start 19 RQAYIRVRM 4.000 position58 DANMLAIYF 3.000 is 20 QAYIRVRMF 3.000 specified, 54 RQWFDANML 3.000the 24 RVRMFFSII 2.400 length 40 VVNDKPISF 2.000 of each 30 SIIFTNEFL1.000 peptide 4 RGNVLSMLI 0.800 is 9 2 LERGNVLSM 0.600 amino 38LTVVNDKPI 0.400 acids, 33 FTNEFLTVV 0.400 the end 52 FNRQWFDAN 0.300position 22 YIRVRMFFS 0.300 for each 14 VEFRDRQAY 0.300 peptide 15EFRDRQAYI 0.240 is the 53 NRQWFDANM 0.200 start 26 RMFFSIIFT 0.200position 57 FDANMLAIY 0.200 plus 6 NVLSMLIEV 0.200 eight 39 TVVNDKPIS0.150 17 RDRQAYIRV 0.120 25 VRMFFSIIF 0.100 31 IIFTNEFLT 0.100 3ERGNVLSML 0.100 21 AYIRVRMFF 0.100 46 ISFKACFNR 0.050 48 FKACFNRQW 0.05023 IRVRMFFSI 0.040 32 IFTNEFLTV 0.030 42 NDKPISFKA 0.030 13 EVEFRDRQA0.030 1 MLERGNVLS 0.030 34 TNEFLTVVN 0.030 55 QWFDANMLA 0.020 10MLIEVEFRD 0.020 9 SMLIEVEFR 0.015 63 AIYFDHRMH 0.015 56 WFDANMLAI 0.01260 NMLAIYFDH 0.010 45 PISFKACFN 0.010 27 MFFSIIFTN 0.010 51 CFNRQWFDA0.010 61 MLAIYFDHR 0.010 43 DKPISFKAC 0.010 37 FLTVVNDKP 0.010 59ANMLAIYFD 0.010 5 GNVLSMLIE 0.010 7 VLSMLIEVE 0.010 50 ACFNRQWFD 0.01011 LIEVEFRDR 0.004 47 SFKACFNRQ 0.003 41 VNDKPISFK 0.003 12 IEVEFRDRQ0.002 36 EFLTVVNDK 0.001 35 NEFLTVVND 0.001 18 DRQAYIRVR 0.001 28FESIIFTNE 0.001 16 FRDRQAYIR 0.000 V2-B35-9mers: 161P5C5 Pos 123456789Score 5 KACFSRQWF 6.000 Portion of 8 FSRQWFDAN 1.500 SEQ ID NO: 6; 9SRQWFDANM 0.200 each start position 2 ISFKACFSR 0.050 is specified, thelength 4 FKACFSRQW 0.050 of each peptide is 6 ACFSRQWFD 0.010 9 aminoacids, 7 CFSRQWFDA 0.010 the end position 1 PISFKACFS 0.010 for eachpeptide 3 SFKACFSRQ 0.003 is the start position plus eight V3-B35-9mers:161P5C5 Pos 123456789 Score 8 LPIYFDHRM 40.000 Portion of 4 DANMLPIYF3.000 SEQ ID NO: 8; 3 FDANMLPIY 0.200 each start position 2 WFDANMLPI0.012 is specified, the length 5 ANMLPIYFD 0.010 of each peptide is 7MLPIYFDHR 0.010 9 amino acids, 6 NMLPIYFDH 0.010 the end position 1QWFDANMLP 0.00 for each peptide 9 PIYFDHRMH 0.002 is the start positionplus eight

TABLE XVIII V1-B35-10mers: 161P5C5 Pos 1234567890 Score 52 FNRQWFDANM6.000 Portion 24 RVRMFFSIIF 6.000 of SEQ ID 29 FSIIFTNEFL 5.000 NO: 4;44 KPISFKACFN 4.000 each 20 QAYIRVRMFF 3.000 start 61 MLAIYFDHRM 2.000position 19 RQAYIRVRMF 2.000 is 22 YIRVRMFFSI 1.200 specified, 39TVVNDKPISF 1.000 the 7 VLSMLIEVEF 1.000 length of 13 EVEFRDRQAY 0.900each 1 MLERGNVLSM 0.600 peptide 37 FLTVVNDKPI 0.400 is 10 31 IIFTNEFLTV0.300 amino 2 LERGNVLSML 0.300 acids, 26 RMFFSIIFTN 0.200 the end 33FTNEFLTVVN 0.200 position 5 GNVLSMLIEV 0.200 for each 54 RQWFDANMLA0.200 peptide 18 DRQAYIRVRM 0.200 is the 47 SFKACFNRQW 0.150 start 38LTVVNDKPIS 0.150 position 53 NRQWFDANML 0.150 plus nine 28 FFSIIFTNEF0.100 30 SIIFTNEFLT 0.100 48 FKACFNRQWF 0.100 43 DKPISFKACF 0.100 57FDANMLAIYF 0.100 9 SMLIEVEFRD 0.010 21 AYIRVRMFFS 0.010 59 ANMLAIYFDH0.010 60 NMLAIYFDHR 0.010 15 EFRDRQAYIR 0.006 17 RDRQAYIRVR 0.006 16FRDRQAYIRV 0.006 34 TNEFLTVVND 0.003 11 LIEVEFRDRQ 0.003 45 PISFKACFNR0.001 27 MFFSIIFTNE 0.001 35 NEFLTVVNDK 0.001 36 EFLTVVNDKP 0.001V2-B35-10mers: 161P5C5 Pos 1234567890 Score 9 FSRQWFDANM 10.000 Portionof 1 KPISFKACFS 0.400 SEQ ID NO: 6; 10 SRQWFDANML 0.400 each startposition 7 ACFSRQWFDA 0.300 is specified, 6 KACFSRQWFD 0.030 the lengthof 3 ISFKACFSRQ 0.010 each peptide is 5 FKACFSRQWF 0.003 10 amino acids,8 CFSRQWFDAN 0.002 the end position 4 SFKACFSRQW 0.002 for each peptide2 PISFKACFSR 0.001 is the start position plus nine V3-B35-10mers:161P5C5 Pos 1234567890 Score 8 MLPIYFDHRM 2.000 Portion of 9 LPIYFDHRMH0.300 SEQ ID NO: 8; 4 FDANMLPIYF 0.100 each start position 2 QWFDANMLPI0.080 is specified, 3 WFDANMLPIY 0.060 the length of 5 DANMLPIYFD 0.030each peptide is 1 RQWFDANMLP 0.020 10 amino acids, 6 ANMLPIYFDH 0.010the end position 7 NMLPIYFDHR 0.010 for each peptide is the startposition plus nine

TABLE XIX Frequently Occurring Motifs avg. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, C2H2 Nucleic acid-bindingprotein type functions as transcription factor, nuclear locationprobable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase,terminal)/b6/petB generate superoxide ig 19% Immunoglobulin domains areone hundred amino domain acids long and include a conserved intradomaindisulfide bond. WD40 18% WD domain, G-beta tandem repeats of about 40repeat residues, each containing a Trp-Asp motif. Function in signaltransduction and protein interaction PDZ 23% PDZ domain may function intargeting signaling molecules to sub- membranous sites LRR 28% LeucineRich short sequence motifs involved Repeat in protein-proteininteractions pkinase 23% Protein kinase conserved catalytic core domaincommon to both serine/threonine and tyrosine protein kinases containingan ATP binding site and a catalytic site PH 16% PH domain pleckstrinhomology involved in intracellular signaling or as constituents of thecytoskeleton EGF 34% EGF-like domain 30-40 amino-acid long found in theextracellular domain of membrane-bound proteins or in secreted proteinsrvt 49% Reverse transcriptase (RNA-dependent DNA polymerase) ank 25% Ankrepeat Cytoplasmic protein, associates integral membrane proteins to thecytoskeleton oxidored_q1 32% NADH- membrane associated. InvolvedUbiquinone/plastoquinone in proton translocation across (complex themembrane I), various chains efhand 24% EF hand calcium-binding domain,consists of a12 residue loop flanked on both sides by a 12 residuealpha-helical domain rvp 79% Retroviral Aspartyl or acid proteases,aspartyl centered on a catalytic protease aspartyl residue Collagen 42%Collagen triple extracellular structural helix repeat (20 proteinsinvolved in copies) formation of connective tissue. The sequenceconsists of the G-X-Y and the polypeptide chains forms a triple helix.fn3 20% Fibronectin type Located in the extracellular III domainligand-binding region of receptors and is about 200 amino acid residueslong with two pairs of cysteines involved in disulfide bonds 7tm_1 19% 7transmembrane seven hydrophobic receptor transmembrane regions, with(rhodopsin the N-terminus located family) extracellularly while the C-terminus is cytoplasmic. Signal through G proteins

TABLE XX MOTIFS AND POST-TRANSLATIONAL MODIFICATIONS OF 161P5C5 Proteinkinase C phosphorylation. 47-49 SfK N-myristoylation site. 5-10 GNvlSM

TABLE XXI PROPERTIES OF 161P5C5 Bioinformatic URL located on the161P5C5-V.1 Program World Wide Web at Outcome ORF ORF finder bp1035-1250 (includes stop codon) Protein length 71 aa Transmembrane TMPred .ch.embnet.org/ one TM aa region 20-40, N terminus inside HMMTop.enzim.hu/hmmtop/ one TM aa 25-41 Sosui .genome.ad.jp/SOSui/ No TM,soluble protein TMHMM .cbs.dtu.dk/services/TMHMM No TM Signal PeptideSignal P .cbs.dtu.dk/services/SignalP/ no pI pI/MW tool.expasy.ch/tools/ pI 7.9 Molecular pI/MW tool .expasy.ch/tools/ 8.6 kDaweight Localization PSORT psort.nibb.ac.jp/ Endoplasmic Reticulum 55%,Lysosome 37% PSORT II psort.nibb.ac.jp/ Cytoplasmic 52%, cytoskeletal21% Motifs Pfam .sanger.ac.uk/Pfam/ no known motif Prints.biochem.ucl.ac.uk/ no known motif Blocks .blocks.fhcrc.org/ WHEP-TRSdomain, Receptor tyrosine kinase class V

TABLE XXII 161P5C5 v.1: HLA Peptide Scoring Results A1 9- mers SYFPEITHIPos 1 2 3 4 5 6 7 8 9 score 14 V E F R D R Q A Y 17 Portion 1 M L E R GN V L S 16 of 56 W F D A N M L A I 16 SEQ ID 57 F D A N M L A I Y 16 NO:4; 11 L I E V E F R D R 13 each 16 F R D R Q A Y I R 13 start 41 V N D KP I S F K 12 position 34 T N E F L T V V N 11 is 13 E V E F R D R Q A 10specified, 32 I F T N E F L T V 9 the 2 L E R G N V L S M 8 length 33 FT N E F L T V V 8 of 37 F L T V V N D K P 7 each 5 G N V L S M L I E 6peptide 38 L T V V N D K P I 6 is 9 42 N D K P I S F K A 6 amino 25 V RM F F S I I F 5 acids, 29 F S I I F T N E F 5 the 40 V V N D K P I S F 5end 47 S F K A C F N R Q 5 position 61 M L A I Y F D H R 5 for 6 N V L SM L I E V 4 each 8 L S M L I E V E F 4 peptide 17 R D R Q A Y I R V 4 is21 A Y I R V R M F F 4 the 26 R M F F S I I F T 4 start 28 F F S I I F TN E 4 position 46 I S F K A C F N R 4 plus 52 F N R Q W F D A N 4 eight58 D A N M L A I Y F 4 3 E R G N V L S M L 3 7 V L S M L I E V E 3 9 S ML I E V E F R 3 10 M L I E V E F R D 3 23 I R V R M F F S I 3 24 R V R MF F S I I 3 30 S I I F T N E F L 3 36 E F L T V V N D K 3 43 D K P I S FK A C 3 55 Q W F D A N M L A 3 4 R G N V L S M L I 2 18 D R Q A Y I R VR 2 20 Q A Y I R V R M F 2 22 Y I R V R M F F S 2 31 I I F T N E F L T 248 F K A C F N R Q W 2 51 C F N R Q W F D A 2 63 A I Y F D H R M H 2 12I E V E F R D R Q 1 19 R Q A Y I R V R M 1 35 N E F L T V V N D 1 49 K AC F N R Q W F 1 50 A C F N R Q W F D 1 54 R Q W F D A N M L 1 59 A N M LA I Y F D 1 161P5C5 v.2: HLA Peptide Scoring Results A1 9- mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 8 F S R Q W F D A N 8 Portion 3 SF K A C F S R Q 5 of 2 I S F K A C F S R 4 SEQ ID 7 C F S R Q W F D A 2NO: 6; 9 S R Q W F D A N M 2 each 4 F K A C F S R Q W 1 start 5 K A C FS R Q W F 1 position 6 A C F S R Q W F D 1 is specified, the length ofeach peptide is 9 amino acids, the end position for each peptide is thestart position plus eight 161P5C5 v.3: HLA Peptide Scoring Results A1 9-mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 2 W F D A N M L P I 16Portion 3 F D A N M L P I Y 16 of 7 M L P I Y F D H R 5 SEQ ID 4 D A N ML P I Y F 4 NO: 8; 6 N M L P I Y F D H 4 each 1 Q W F D A N M L P 3start 5 A N M L P I Y F D 2 position 9 P I Y F D H R M H 1 is specified,the length of each peptide is 9 amino acids, the end position for eachpeptide is the start position plus eight

TABLE XXIII 161P5C5 v.1: HLA Peptide Scoring Results A*0201 9-mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 33 F T N E F L T V V 23 Portion 6N V L S M L I E V 22 of 30 S I I F T N E F L 21 SEQ ID 7 V L S M L I E VE 16 NO: 4; 9 S M L I E V E F R 16 each 26 R M F F S I I F T 16 start 2L E R G N V L S M 15 position 31 I I F T N E F L T 15 is 10 M L I E V EF R D 14 specified, 32 I F T N E F L T V 14 the 1 M L E R G N V L S 13length 24 R V R M F F S I I 13 of 38 L T V V N D K P I 13 each 61 M L AI Y F D H R 13 peptide 62 L A I Y F D H R M 13 is 9 23 I R V R M F F S I12 amino 37 F L T V V N D K P 12 acids, 3 E R G N V L S M L 11 the 54 RQ W F D A N M L 11 end 4 R G N V L S M L I 10 position 17 R D R Q A Y IR V 10 for 22 Y I R V R M F F S 10 each 40 V V N D K P I S F 10 peptide56 W F D A N M L A I 10 is 60 N M L A I L F D H 10 the 63 A I Y F D H RM H 10 start 11 L I E V E F R D R 9 position 20 Q A Y I R V R M F 9 plus29 F S I I F T N E F 9 eight 57 F D A N M L A I Y 9 15 E F R D R Q A Y I8 59 A N M L A I Y F D 8 8 L S M L I E V E F 7 19 R Q A Y I R V R M 7 27M F F S I I F T N 7 35 N E F L T V V N D 7 42 N D K P I S F K A 7 53 N RQ W F D A N M 7 41 V N D K P I S F K 6 44 K P I S F K A C F 6 55 Q W F DA N M L A 6 12 I E V E F R D R Q 5 39 T V V N D K P I S 5 45 P I S F K AC F N 5 51 C F N R Q W F D A 5 58 D A N M L A I Y F 5 18 D R Q A Y I R VR 4 21 A Y I R V R M F F 4 36 E F L T V V N D K 4 48 F K A C F N R Q W 449 K A C F N R Q W F 4 13 E V E F R D R Q A 3 14 V E F R D R Q A Y 3 34T N E F L T V V N 3 46 I S F K A C F N R 3 50 A C F N R Q W F D 3 5 G NV L S M L I E 2 16 F R D R Q A Y I R 2 25 V R M F F S I I F 2 47 S F K AC F N R Q 2 52 F N R Q W F D A N 2 28 F F S I I F T N E 1 43 D K P I S FK A C −1 161P5C5 v.2: HLA Peptide Scoring Results A*0201 9-mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 9 S R Q W F D A N M 9 Portion 1 PI S F K A C F S 5 of 2 I S F K A C F S R 5 SEQ ID 4 F K A C F S R Q W 4NO: 6; 5 K A C F S R Q W F 4 each 7 C F S R Q W F D A 4 start 6 A C F SR Q W F D 3 position 8 F S R Q W F D A N 2 is 3 S F K A C F S R Q 1specified, the length of each peptide is 9 amino acids, the end positionfor each peptide is the start position plus eight 161P5C5 v.3: HLAPeptide Scoring Results A*0201 9-mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9score 6 N M L P I Y F D H 12 Portion 7 M L P I Y F D H R 11 of 2 W F D AN M L P I 9 SEQ ID 5 A N M L P I Y F D 9 NO: 8; 8 L P I Y F D H R M 9each 3 F D A N M L P I Y 8 start 4 D A N M L P I Y F 5 position 9 P I YF D H R M H 5 is 1 Q W F D A N M L P 2 specified, the length of eachpeptide is 9 amino acids, the end position for each peptide is the startposition plus eight

TABLE XXIV 161P5C5: HLA Peptide Scoring Results A*0202 9-mers SYFPEITHIPos 1 2 3 4 5 6 7 8 9 score . NO DATA

TABLE XXV 161P5C5: HLA Peptide Scoring Results A*0203 9-mers SYFPEITHIPos 1 2 3 4 5 6 7 8 9 score . NO DATA

TABLE XXVI 161P5C5 v.1: HLA Peptide Scoring Results A3 9-mers SYFPEITHIPos 1 2 3 4 5 6 7 8 9 score 24 R V R M F F S I I 20 Portion 63 A I Y F DH R M H 20 of SEQ 1 M L E R G N V L S 19 ID NO: 4; 40 V V N D K P I S F19 each 21 A Y I R V R M F F 16 start 61 M L A I Y F D H R 16 position10 M L I E V E F R D 15 is 13 E V E F R D R Q A 15 specified, 7 V L S ML I E V E 14 the 20 Q A Y I R V R M F 14 length 22 Y I R V R M F F S 13of each 36 E F L T V V N D K 13 peptide 41 V N D K P I S F K 13 is 9 44K P I S F K A C F 13 amino 6 N V L S M L I E V 12 acids, 11 L I E V E FR D R 12 the end 30 S I I F T N E F L 12 position 45 P I S F K A C F N12 for each 14 V E F R D R Q A Y 11 peptide 37 F L T V V N D K P 11 isthe 39 T V V N D K P I S 11 start 57 F D A N M L A I Y 11 position 2 L ER G N V L S M 10 plus 16 F R D R Q A Y I R 10 eight 18 D R Q A Y I R V R10 19 R Q A Y I R V R M 10 31 I I F T N E F L T 10 32 I F T N E F L T V10 8 L S M L I E V E F 9 15 E F R D R Q A Y I 9 49 K A C F N R Q W F 9 9S M L I E V E F R 8 29 F S I I F T N E F 8 34 T N E F L T V V N 8 54 R QW F D A N M L 8 60 N M L A I Y F D H 8 17 R D R Q A Y I R V 7 23 I R V RM F F S I 7 58 D A N M L A I Y F 7 3 E R G N V L S M L 6 33 F T N E F LT V V 6 46 I S F K A C F N R 6 50 A C F N R Q W F D 6 56 W F D A N M L AI 6 4 R G N V L S M L I 5 35 N E F L T V V N D 5 5 G N V L S M L I E 412 I E V E F R D R Q 4 25 V R M F F S I I F 4 26 R M F F S I I F T 4 27M F F S I I F T N 4 42 N D K P I S F K A 4 47 S F K A C F N R Q 4 48 F KA C F N R Q W 4 52 F N R Q W F D A N 4 55 Q W F D A N M L A 4 59 A N M LA I Y F D 4 51 C F N R Q W F D A 3 53 N R Q W F D A N M 3 62 L A I Y F DH R M 3 28 F F S I I F T N E 2 43 D K P I S F K A C 2 161P5C5 v.2: HLAPeptide Scoring Results A3 9-mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score1 P I S F K A C F S 12 Portion 5 K A C F S R Q W F 9 of SEQ 2 I S F K AC F S R 8 ID NO: 6; 3 S F K A C F S R Q 7 each 6 A C F S R Q W F D 5start 4 F K A C F S R Q W 4 position 8 F S R Q W F D A N 4 is 9 S R Q WF D A N M 4 specified, 7 C F S R Q W F D A 3 the length of each peptideis 9 amino acids, the end position for each peptide is the startposition plus eight 161P5C5 v.3: HLA Peptide Scoring Results A3 9- mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 9 P I Y F D H R M H 17 Portion 7 ML P I Y F D H R 15 of SEQ 6 N M L P I Y F D H 9 ID 3 F D A N M L P I Y 8NO: 8; 2 W F D A N M L P I 5 each 4 D A N M L P I Y F 5 start 1 Q W F DA N M L P 4 position 5 A N M L P I Y F D 4 is 8 L P I Y F D H R M 3specified, the length of each peptide is 9 amino acids, the end positionfor each peptide is the start position plus eight

TABLE XXVII 161P5C5 v.1: HLA Peptide Scoring Results A26 9- mersSYFPEITHI Pos 123456789 score 40 VVNDKPISF 23 Portion 3 ERGNVLSML 20 ofSEQ 30 SIIFTNEFL 20 ID NO: 4; 13 EVEFRDRQA 17 each 58 DANMLAIYF 17 start6 NVLSMLIEV 16 position 33 FTNEFLTVV 16 is 36 EFLTVVNDK 16 specified, 57FDANMLAIY 16 the 2 LERGNVLSM 15 length 20 QAYIRVRMF 15 of each 24RVRMFFSII 15 peptide 11 LIEVEFRDR 14 is 9 14 VEFRDRQAY 14 amino 15EFRDRQAYI 14 acids, 22 YIRVRMFFS 14 the end 44 KPISFKACF 14 position 61MLAIYFDHR 14 for each 7 VLSMLIEVE 13 peptide 10 MLIEVEFRD 13 is the 21AYIRVRMFF 13 start 27 MFFSIIFTN 13 position 29 FSIIFTNEF 13 plus 43DKPISFKAC 13 eight 8 LSMLIEVEF 12 31 IIFTNEFLT 12 47 SFKACFNRQ 12 56WFDANMLAI 12 19 RQAYIRVRM 11 28 FFSIIFTNE 11 39 TVVNDKPIS 11 45PISFKACFN 11 49 KACFNRQWF 11 62 LAIYFDHRM 11 63 AIYFDHRMH 11 18DRQAYIRVR 10 25 VRMFFSIIF 10 32 IFTNEFLTV 10 38 LTVVNDKPI 10 53NRQWFDANM 10 1 MLERGNVLS 9 37 FLTVVNDKP 9 54 RQWFDANML 9 35 NEFLTVVND 826 RMFFSIIFT 7 41 VNDKPISFK 7 51 CFNRQWFDA 7 17 RDRQAYIRV 6 52 FNRQWFDAN6 5 GNVLSMLIE 5 23 IRVRMFFSI 5 9 SMLIEVEFR 4 42 NDKPISFKA 4 46 ISFKACFNR4 55 QWFDANMLA 4 59 ANMLAIYFD 4 60 NMLAIYFDH 4 12 IEVEFRDRQ 3 4RGNVLSMLI 2 16 FRDRQAYIR 2 34 TNEFLTVVN 2 48 FKACFNRQW 2 50 ACFNRQWFD 2161P5C5 v.2: HLA Peptide Scoring Results A26 9-mers SYFPEITHI Pos 1 2 34 5 6 7 8 9 score 3 S F K A C F S R Q 13 Portion 5 K A C F S R Q W F 12of SEQ 1 P I S F K A C F S 11 ID 9 S R Q W F D A N M 10 NO: 6; 7 C F S RQ W F D A 7 each 8 F S R Q W F D A N 6 start 2 I S F K A C F S R 4position 6 A C F S R Q W F D 3 is 4 F K A C F S R Q W 2 specified, thelength of each peptide is 9 amino acids, the end position for eachpeptide is the start position plus eight 161P5C5 v.3: HLA PeptideScoring Results A26 9- mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 4 D AN M L P I Y F 17 Portion 3 F D A N M L P I Y 16 of 7 M L P I Y F D H R14 SEQ ID 2 W F D A N M L P I 11 NO: 8; 8 L P I Y F D H R M 11 each 9 PI Y F D H R M H 11 start 6 N M L P I Y F D H 6 position 1 Q W F D A N ML P 4 is 5 A N M L P I Y F D 4 specified, the length of each peptide is9 amino acids, the end position for each peptide is the start positionplus eight

TABLE XXVIII v.1: HLA Peptide Scoring Results B*0702 9-mers SYFPEITHIPos 1 2 3 4 5 6 7 8 9 score 44 K P I S F K A C F 18 Portion 2 L E R G NV L S M 13 of 3 E R G N V L S M L 12 SEQ ID 19 R Q A Y I R V R M 11 NO:4; 30 S I I F T N E F L 11 each 54 R Q W F D A N M L 11 start 56 W F D AN M L A I 11 position 15 E F R D R Q A Y I 10 is 21 A Y I R V R M F F 10specified, 24 R V R M F F S I I 10 the 32 I F T N E F L T V 10 length 8L S M L I E V E F 9 of 17 R D R Q A Y I R V 9 each 23 I R V R M F F S I8 peptide 26 R M F F S I I F T 8 is 9 33 F T N E F L T V V 8 amino 49 KA C F N R Q W F 8 acids, 4 R G N V L S M L I 7 the 13 E V E F R D R Q A7 end 20 Q A Y I R V R M F 7 position 25 V R M F F S I I F 7 for 29 F SI I F T N E F 7 each 31 I I F T N E F L T 7 peptide 38 L T V V N D K P I7 is 42 N D K P I S F K A 7 the 53 N R Q W F D A N M 7 start 6 N V L S ML I E V 6 position 40 V V N D K P I S F 6 plus 41 V N D K P I S F K 6eight 51 C F N R Q W F D A 6 55 Q W F D A N M L A 6 58 D A N M L A I Y F6 62 L A I Y F D H R M 6 28 F F S I I F T N E 4 34 T N E F L T V V N 452 F N R Q W F D A N 4 59 A N M L A I Y F D 4 7 V L S M L I E V E 3 22 YI R V R M F F S 3 35 N E F L T V V N D 3 45 P I S F K A C F N 3 50 A C FN R Q W F D 3 1 M L E R G N V L S 2 5 G N V L S M L I E 2 9 S M L I E VE F R 2 12 I E V E F R D R Q 2 14 V E F R D R Q A Y 2 18 D R Q A Y I R VR 2 36 E F L T V V N D K 2 43 D K P I S F K A C 2 46 I S F K A C F N R 247 S F K A C F N R Q 2 57 F D A N M L A I Y 2 61 M L A I Y F D H R 2 63A I Y F D H R M H 2 11 L I E V E F R D R 1 16 F R D R Q A Y I R 1 39 T VV N D K P I S 1 48 F K A C F N R Q W 1 60 N M L A I Y F D H 1 v.2: HLAPeptide Scoring Results B*0702 9-mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9score 5 K A C F S R Q W F 8 Portion 7 C F S R Q W F D A 8 of 9 S R Q W FD A N M 7 SEQ ID 8 F S R Q W F D A N 4 NO: 6; 1 P I S F K A C F S 3 each6 A C F S R Q W F D 3 start 2 I S F K A C F S R 2 position 3 S F K A C FS R Q 2 is 4 F K A C F S R Q W 1 specified, the length of each peptideis 9 amino acids, the end position for each peptide is the startposition plus eight v.3: HLA Peptide Scoring Results B*0702 9-mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 8 L P I Y F D H R M 16 Portion 2 WF D A N M L P I 11 of 5 A N M L P I Y F D 7 SEQ ID 4 D A N M L P I Y F 6NO: 8; 3 F D A N M L P I Y 2 each 6 N M L P I Y F D H 1 start 7 M L P IY F D H R 1 position is specified, the length of each peptide is 9 aminoacids, the end position for each peptide is the start position pluseight

TABLE XXIX 161P5C5 v.1: HLA Peptide Scoring Results B*08 9- mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 15 E F R D R Q A Y I 22 Portion 20Q A Y I R V R M F 19 of 22 Y I R V R M F F S 19 SEQ ID 30 S I I F T N EF L 17 NO: 4; 40 V V N D K P I S F 17 each 45 P I S F K A C F N 14 start3 E R G N V L S M L 12 position 24 R V R M F F S I I 12 is 44 K P I S FK A C F 12 specified, 47 S F K A C F N R Q 12 the 13 E V E F R D R Q A10 length 42 N D K P I S F K A 10 of 49 K A C F N R Q W F 10 each 54 R QW F D A N M L 10 peptide 58 D A N M L A I Y F 10 is 9 8 L S M L I E V EF 9 amino 37 F L T V V N D K P 9 acids, 1 M L E R G N V L S 8 the 7 V LS M L I E V E 8 end 10 M L I E V E F R D 8 position 29 F S I I F T N E F8 for 38 L T V V N D K P I 8 each 50 A C F N R Q W F D 8 peptide 52 F NR Q W F D A N 8 is 2 L E R G N V L S M 7 the 4 R G N V L S M L I 7 start23 I R V R M F F S I 7 position 25 V R M F F S I I F 7 plus 56 W F D A NM L A I 7 eight 17 R D R Q A Y I R V 6 21 A Y I R V R M F F 6 61 M L A IY F D H R 6 11 L I E V E F R D R 5 31 I I F T N E F L T 5 62 L A I Y F DH R M 5 63 A I Y F D H R M H 4 9 S M L I E V E F R 3 12 I E V E F R D RQ 3 33 F T N E F L T V V 3 35 N E F L T V V N D 3 36 E F L T V V N D K 35 G N V L S M L I E 2 14 V E F R D R Q A Y 2 19 R Q A Y I R V R M 2 41 VN D K P I S F K 2 43 D K P I S F K A C 2 48 F K A C F N R Q W 2 59 A N ML A I Y F D 2 6 N V L S M L I E V 1 16 F R D R Q A Y I R 1 18 D R Q A YI R V R 1 26 R M F F S I I F T 1 27 M F F S I I F T N 1 28 F F S I I F TN E 1 32 I F T N E F L T V 1 34 T N E F L T V V N 1 46 I S F K A C F N R1 55 Q W F D A N M L A 1 57 F D A N M L A I Y 1 161P5C5 v.2: HLA PeptideScoring Results B*08 9- mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 1 P IS F K A C F S 14 Portion 3 S F K A C F S R Q 12 of 5 K A C F S R Q W F10 SEQ ID 6 A C F S R Q W F D 8 NO: 6; 8 F S R Q W F D A N 8 each 9 S RQ W F D A N M 2 start 2 I S F K A C F S R 1 position 4 F K A C F S R Q W1 is specified, the length of each peptide is 9 amino acids, the endposition for each peptide is the start position plus eight 161P5C5 v.3:HLA Peptide Scoring Results B*08 9- mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9score 4 D A N M L P I Y F 10 Portion 8 L P I Y F D H R M 7 of 2 W F D AN M L P I 6 SEQ ID 7 M L P I Y F D H R 6 NO: 8; 9 P I Y F D H R M H 4each 5 A N M L P I Y F D 2 start 1 Q W F D A N M L P 1 position 3 F D AN M L P I Y 1 is specified, the length of each peptide is 9 amino acids,the end position for each peptide is the start position plus eight

TABLE XXX 161P5C5 v.1: HLA Peptide Scoring Results B*1510 9-mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 19 R Q A Y I R V R M 12 Portion 3E R G N V L S M L 11 of 30 S I I F T N E F L 10 SEQ ID 54 R Q W F D A NM L 10 NO: 4; 2 L E R G N V L S M 9 each 8 L S M L I E V E F 9 start 20Q A Y I R V R M F 9 position 40 V V N D K P I S F 8 is 49 K A C F N R QW F 8 specified, 62 L A I Y F D H R M 8 the 21 A Y I R V R M F F 7length 29 F S I I F T N E F 7 of 44 K P I S F K A C F 7 each 12 I E V EF R D R Q 6 peptide 25 V R M F F S I I F 6 is 9 34 T N E F L T V V N 6amino 53 N R Q W F D A N M 6 acids, 58 D A N M L A I Y F 6 the 18 D R QA Y I R V R 5 end 1 M L E R G N V L S 4 position 7 V L S M L I E V E 4for 11 L I E V E F R D R 4 each 13 E V E F R D R Q A 4 peptide 10 M L IE V E F R D 3 is 17 R D R Q A Y I R V 3 the 22 Y I R V R M F F S 3 start31 I I F T N E F L T 3 position 32 I F T N E F L T V 3 plus 33 F T N E FL T V V 3 eight 35 N E F L T V V N D 3 39 T V V N D K P I S 3 41 V N D KP I S F K 3 46 I S F K A C F N R 3 48 F K A C F N R Q W 3 56 W F D A N ML A I 3 5 G N V L S M L I E 2 14 V E F R D R Q A Y 2 15 E F R D R Q A YI 2 23 I R V R M F F S I 2 27 M F F S I I F T N 2 36 E F L T V V N D K 238 L T V V N D K P I 2 42 N D K P I S F K A 2 47 S F K A C F N R Q 2 52F N R Q W F D A N 2 63 A I Y F D H R M H 2 4 R G N V L S M L I 1 6 N V LS M L I E V 1 9 S M L I E V E F R 1 26 R M F F S I I F T 1 28 F F S I IF T N E 1 37 F L T V V N D K P 1 43 D K P I S F K A C 1 45 P I S F K A CF N 1 55 Q W F D A N M L A 1 57 F D A N M L A I Y 1 59 A N M L A I Y F D1 61 M L A I Y F D H R 1 161P5C5 v.2: HLA Peptide Scoring Results B*15109-mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 5 K A C F S R Q W F 7Portion 9 S R Q W F D A N M 6 of 2 I S F K A C F S R 3 SEQ ID 4 F K A CF S R Q W 3 NO: 6; 3 S F K A C F S R Q 2 each 8 F S R Q W F D A N 2start 1 P I S F K A C F S 1 position 7 C F S R Q W F D A 1 is specified,the length of each peptide is 9 amino acids, the end position for eachpeptide is the start position plus eight 161P5C5 v.3: HLA PeptideScoring Results B*1510 9-mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 8 LP I Y F D H R M 8 Portion 4 D A N M L P I Y F 7 of 2 W F D A N M L P I 2SEQ ID 3 F D A N M L P I Y 2 NO: 8; 5 A N M L P I Y F D 2 each 6 N M L PI Y F D H 2 start 9 P I Y F D H R M H 2 position 1 Q W F D A N M L P 1is specified, the length of each peptide is 9 amino acids, the endposition for each peptide is the start position plus eight

TABLE XXXI 161P5C5 v.1: HLA Peptide Scoring Results B*2705 9-mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 18 D R Q A Y I R V R 24 Portion 3E R G N V L S M L 23 of 16 F R D R Q A Y I R 22 SEQ ID 25 V R M F F S II F 22 NO: 4; 53 N R Q W F D A N M 22 each 23 I R V R M F F S I 19 start46 I S F K A C F N R 18 position 41 V N D K P I S F K 17 is 19 R Q A Y IR V R M 16 specified, 44 K P I S F K A C F 16 the 54 R Q W F D A N M L16 length 21 A Y I R V R M F F 15 of 36 E F L T V V N D K 15 each 8 L SM L I E V E F 14 peptide 9 S M L I E V E F R 14 is 9 29 F S I I F T N EF 14 amino 2 L E R G N V L S M 13 acids, 4 R G N V L S M L I 13 the 20 QA Y I R V R M F 13 end 30 S I I F T N E F L 13 position 40 V V N D K P IS F 13 for 58 D A N M L A I Y F 13 each 62 L A I Y F D H R M 13 peptide26 R M F F S I I F T 12 is 49 K A C F N R Q W F 12 the 11 L I E V E F RD R 11 start 14 V E F R D R Q A Y 11 position 24 R V R M F F S I I 11plus 60 N M L A I Y F D H 11 eight 63 A I Y F D H R M H 11 57 F D A N ML A I Y 10 61 M L A I Y F D H R 10 15 E F R D R Q A Y I 9 17 R D R Q A YI R V 9 27 M F F S I I F T N 8 35 N E F L T V V N D 8 38 L T V V N D K PI 8 5 G N V L S M L I E 7 50 A C F N R Q W F D 7 56 W F D A N M L A I 710 M L I E V E F R D 6 59 A N M L A I Y F D 6 6 N V L S M L I E V 5 7 VL S M L I E V E 5 12 I E V E F R D R Q 5 28 F F S I I F T N E 5 33 F T NE F L T V V 5 42 N D K P I S F K A 5 55 Q W F D A N M L A 5 1 M L E R GN V L S 4 31 I I F T N E F L T 4 32 I F T N E F L T V 4 37 F L T V V N DK P 4 34 T N E F L T V V N 3 39 T V V N D K P I S 3 47 S F K A C F N R Q3 13 E V E F R D R Q A 2 45 P I S F K A C F N 2 48 F K A C F N R Q W 252 F N R Q W F D A N 2 43 D K P I S F K A C 1 161P5C5 v.2: HLA PeptideScoring Results B*2705 9-mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 9 SR Q W F D A N M 22 Portion 2 I S F K A C F S R 17 of 5 K A C F S R Q W F13 SEQ ID 6 A C F S R Q W F D 7 NO: 6; 3 S F K A C F S R Q 3 each 1 P IS F K A C F S 2 start 4 F K A C F S R Q W 2 position 8 F S R Q W F D A N2 is specified, the length of each peptide is 9 amino acids, the endposition for each peptide is the start position plus eight v.3: HLAPeptide Scoring Results B*2705 9-mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9score 8 L P I Y F D H R M 13 Portion 4 D A N M L P I Y F 12 of 6 N M L PI Y F D H 12 SEQ ID 3 F D A N M L P I Y 10 NO: 8; 7 M L P I Y F D H R 10each 9 P I Y F D H R M H 10 start 2 W F D A N M L P I 7 position 5 A N ML P I Y F D 7 is 1 Q W F D A N M L P 5 specified, the length of eachpeptide is 9 amino acids, the end position for each peptide is the startposition plus eight

TABLE XXXII 161P5C5 v.1: HLA Peptide Scoring Results B*2709 9-mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 3 E R G N V L S M L 20 Portion 23I R V R M F F S I 20 of 53 N R Q W F D A N M 19 SEQ ID 25 V R M F F S II F 18 NO: 4; 54 R Q W F D A N M L 16 each 19 R Q A Y I R V R M 15 start17 R D R Q A Y I R V 13 position 4 R G N V L S M L I 12 is 16 F R D R QA Y I R 12 specified, 24 R V R M F F S I I 12 the 44 K P I S F K A C F12 length 6 N V L S M L I E V 11 of 20 Q A Y I R V R M F 11 each 30 S II F T N E F L 11 peptide 32 I F T N E F L T V 11 is 9 2 L E R G N V L SM 10 amino 8 L S M L I E V E F 10 acids, 18 D R Q A Y I R V R 10 the 21A Y I R V R M F F 10 end 49 K A C F N R Q W F 10 position 62 L A I Y F DH R M 10 for 29 F S I I F T N E F 9 each 38 L T V V N D K P I 9 peptide56 W F D A N M L A I 9 is 58 D A N M L A I Y F 9 the 15 E F R D R Q A YI 8 start 33 F T N E F L T V V 8 position 40 V V N D K P I S F 8 plus 26R M F F S I I F T 6 eight 5 G N V L S M L I E 5 35 N E F L T V V N D 527 M F F S I I F T N 4 46 I S F K A C F N R 4 63 A I Y F D H R M H 4 9 SM L I E V E F R 3 12 I E V E F R D R Q 3 14 V E F R D R Q A Y 3 31 I I FT N E F L T 3 36 E F L T V V N D K 3 39 T V V N D K P I S 3 50 A C F N RQ W F D 3 60 N M L A I Y F D H 3 10 M L I E V E F R D 2 28 F F S I I F TN E 2 42 N D K P I S F K A 2 55 Q W F D A N M L A 2 59 A N M L A I Y F D2 1 M L E R G N V L S 1 13 E V E F R D R Q A 1 34 T N E F L T V V N 1 37F L T V V N D K P 1 45 P I S F K A C F N 1 47 S F K A C F N R Q 1 48 F KA C F N R Q W 1 161P5C5 v.2: HLA Peptide Scoring Results B*2709 9- mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 9 S R Q W F D A N M 19 Portion 5 KA C F S R Q W F 10 of SEQ 6 A C F S R Q W F D 4 ID 2 I S F K A C F S R 3NO: 6; 1 P I S F K A C F S 1 each 3 S F K A C F S R Q 1 start 4 F K A CF S R Q W 1 position is specified, the length of each peptide is 9 aminoacids, the end position for each peptide is the start position pluseight 161P5C5 v.3: HLA Peptide Scoring Results B*2709 9-mers SYFPEITHIPos 1 2 3 4 5 6 7 8 9 score 8 L P I Y F D H R M 10 Portion 2 W F D A N ML P I 9 of 4 D A N M L P I Y F 8 SEQ ID 6 N M L P I Y F D H 4 NO: 8; 9 PI Y F D H R M H 3 each 1 Q W F D A N M L P 2 start 5 A N M L P I Y F D 2position 3 F D A N M L P I Y 1 is specified, the length of each peptideis 9 amino acids, the end position for each peptide is the startposition plus eight

TABLE XXXIII 161P5C5 v.1: HLA Peptide Scoring Results B*4402 9-mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 14 V E F R D R Q A Y 26 Portion 21A Y I R V R M F F 18 of SEQ 35 N E F L T V V N D 16 ID NO: 4; 29 F S I IF T N E F 15 each 2 L E R G N V L S M 14 start 3 E R G N V L S M L 14position 8 L S M L I E V E F 14 is 30 S I I F T N E F L 14 specified, 44K P I S F K A C F 14 the 20 Q A Y I R V R M F 13 length 40 V V N D K P IS F 13 of each 49 K A C F N R Q W F 13 peptide 56 W F D A N M L A I 13is 9 15 E F R D R Q A Y I 12 amino 25 V R M F F S I I F 12 acids, 48 F KA C F N R Q W 12 the end 54 R Q W F D A N M L 12 position 57 F D A N M LA I Y 12 for each 58 D A N M L A I Y F 12 peptide 12 I E V E F R D R Q11 is the 38 L T V V N D K P I 11 start 23 I R V R M F F S I 10 position4 R G N V L S M L I 9 plus 24 R V R M F F S I I 9 eight 59 A N M L A I YF D 8 27 M F F S I I F T N 7 50 A C F N R Q W F D 7 26 R M F F S I I F T6 36 E F L T V V N D K 6 42 N D K P I S F K A 6 43 D K P I S F K A C 6 7V L S M L I E V E 5 13 E V E F R D R Q A 5 18 D R Q A Y I R V R 5 31 I IF T N E F L T 5 6 N V L S M L I E V 4 32 I F T N E F L T V 4 41 V N D KP I S F K 4 55 Q W F D A N M L A 4 62 L A I Y F D H R M 4 63 A I Y F D HR M H 4 1 M L E R G N V L S 3 9 S M L I E V E F R 3 10 M L I E V E F R D3 16 F R D R Q A Y I R 3 19 R Q A Y I R V R M 3 28 F F S I I F T N E 334 T N E F L T V V N 3 47 S F K A C F N R Q 3 52 F N R Q W F D A N 3 60N M L A I Y F D H 3 5 G N V L S M L I E 2 11 L I E V E F R D R 2 33 F TN E F L T V V 2 45 P I S F K A C F N 2 46 I S F K A C F N R 2 17 R D R QA Y I R V 1 22 Y I R V R M F F S 1 37 F L T V V N D K P 1 39 T V V N D KP I S 1 51 C F N R Q W F D A 1 53 N R Q W F D A N M 1 61 M L A I Y F D HR 1 161P5C5 v.2: HLA Peptide Scoring Results B*4402 9-mers SYFPEITHI Pos1 2 3 4 5 6 7 8 9 score 4 F K A C F S R Q W 12 Portion 5 K A C F S R Q WF 12 of 6 A C F S R Q W F D 6 SEQ ID 8 F S R Q W F D A N 3 NO: 6; 1 P IS F K A C F S 2 each 2 I S F K A C F S R 2 start 3 S F K A C F S R Q 2position 7 C F S R Q W F D A 2 is 9 S R Q W F D A N M 1 specified, thelength of each peptide is 9 amino acids, the end position for eachpeptide is the start position plus eight v.3: HLA Peptide ScoringResults B*4402 9-mers SYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 3 F D A N ML P I Y 12 Portion 4 D A N M L P I Y F 12 of 2 W F D A N M L P I 11 SEQID 5 A N M L P I Y F D 8 NO: 8; 6 N M L P I Y F D H 6 each 1 Q W F D A NM L P 4 start 8 L P I Y F D H R M 4 position 7 M L P I Y F D H R 2 is 9P I Y F D H R M H 1 specified, the length of each peptide is 9 aminoacids, the end position for each peptide is the start position pluseight

TABLE XXXIV 161P5C5 v.1: HLA Peptide Scoring Results B*5101 9-mersSYFPEITHI Pos 1 2 3 4 5 6 7 8 9 score 20 Q A Y I R V R M F 17 Portion 4R G N V L S M L I 16 of 33 F T N E F L T V V 16 SEQ ID 58 D A N M L A IY F 16 NO: 4; 6 N V L S M L I E V 14 each 23 I R V R M F F S I 14 start32 I F T N E F L T V 14 position 38 L T V V N D K P I 13 is 62 L A I Y FD H R M 13 specified, 56 W F D A N M L A I 12 the 15 E F R D R Q A Y I11 length 18 D R Q A Y I R V R 11 of 24 R V R M F F S I I 11 each 17 R DR Q A Y I R V 10 peptide 44 K P I S F K A C F 10 is 9 49 K A C F N R Q WF 10 amino 3 E R G N V L S M L 9 acids, 54 R Q W F D A N M L 9 the 30 SI I F T N E F L 8 end 36 E F L T V V N D K 8 position 43 D K P I S F K AC 8 for 2 L E R G N V L S M 6 each 10 M L I E V E F R D 6 peptide 35 N EF L T V V N D 6 is 37 F L T V V N D K P 6 the 7 V L S M L I E V E 5start 12 I E V E F R D R Q 5 position 27 M F F S I I F T N 5 on 34 T N EF L T V V N 5 plus 40 V V N D K P I S F 5 eight 63 A I Y F D H R M H 5 9S M L I E V E F R 4 11 L I E V E F R D R 4 26 R M F F S I I F T 4 31 I IF T N E F L T 4 46 I S F K A C F N R 4 47 S F K A C F N R Q 4 60 N M L AI Y F D H 4 61 M L A I Y F D H R 4 1 M L E R G N V L S 3 8 L S M L I E VE F 3 28 F F S I I F T N E 3 41 V N D K P I S F K 3 42 N D K P I S F K A3 48 F K A C F N R Q W 3 55 Q W F D A N M L A 3 57 F D A N M L A I Y 3 5G N V L S M L I E 2 14 V E F R D R Q A Y 2 16 F R D R Q A Y I R 2 19 R QA Y I R V R M 2 21 A Y I R V R M F F 2 22 Y I R V R M F F S 2 25 V R M FF S I I F 2 29 F S I I F T N E F 2 45 P I S F K A C F N 2 52 F N R Q W FD A N 2 13 E V E F R D R Q A 1 39 T V V N D K P I S 1 50

TABLE XXXV 161P5C5 v.1: HLA Peptide Scoring Results A1 10- mersSYFPEITHI Pos 1234567890 score 13 EV EFRDRQAY 26 Portion of 56 WFDANMLAIY 25 SEQ ID NO: 4; 1 ML ERGNVLSM 18 each start 16 FR DRQAYIRV 15position is 41 VN DKPISFKA 13 specified, 11 LI EVEFRDRQ 11 the length 34TN EFLTVVND 11 of each 31 II FTNEFLTV 9 peptide is 33 FT NEFLTVVN 8 10amino 42 ND KPISFKAC 7 acids, the 46 IS FKACFNRQ 7 end position 4 RGNVLSMLIE 6 for each 29 FS IIFTNEFL 6 peptide is 38 LT VVNDKPIS 6 thestart 55 QW FDANMLAI 6 position 8 LS MLIEVEFR 5 plus nine 22 YI RVRMFFSI5 25 VR MFFSIIFT 5 57 FD ANMLAIYF 5 5 GN VLSMLIEV 4 10 ML IEVEFRDR 4 24RV RMFFSIIF 4 30 SI IFTNEFLT 4 39 TV VNDKPISF 4 23 IR VRMFFSII 3 27 MFFSIIFTNE 3 36 EF LTVVNDKP 3 47 SF KACFNRQW 3 51 CF NRQWFDAN 3 54 RQWFDANMLA 3 60 NM LAIYFDHR 3 2 LE RGNVLSML 2 3 ER GNVLSMLI 2 7 VLSMLIEVEF 2 9 SM LIEVEFRD 2 19 RQ AYIRVRMF 2 37 FL TVVNDKPI 2 48 FKACFNRQWF 2 6 NV LSMLIEVE 1 14 VE FRDRQAYI 1 17 RD RQAYIRVR 1 18 DRQAYIRVRM 1 20 QA YIRVRMFF 1 21 AY IRVRMFFS 1 28 FF SIIFTNEF 1 32 IFTNEFLTVV 1 35 NE FLTVVNDK 1 40 VV NDKPISFK 1 50 AC FNRQWFDA 1 52 FNRQWFDANM 1 53 NR QWFDANML 1 59 AN MLAIYFDH 1 61 ML AIYFDHRM 1 62 LAIYFDHRMH 1 161P5C5 v.2: HLA Peptide Scoring Results A1 10- mersSYFPEITHI Pos 1234567890 score 3 IS FKACFSRQ 7 Portion 9 FS RQWFDANM 5of SEQ 8 CF SRQWFDAN 3 ID NO: 6; 10 SR QWFDANML 3 each 4 SF KACFSRQW 2start 5 FK ACFSRQWF 2 position 7 AC FSRQWFDA 1 is specified, the lengthof each peptide is 10 amino acids, the end position for each peptide isthe start position plus nine 161P5C5 v.3: HLA Peptide Scoring Results A110- mers SYFPEITHI Pos 1234567890 score 3 WF DANMLPIY 25 Portion 7 NMLPIYFDHR 7 of 2 QW FDANMLPI 6 SEQ ID 4 FD ANMLPIYF 5 NO: 8; 1 RQWFDANMLP 3 each 6 AN MLPIYFDH 1 start 8 ML PIYFDHRM 1 position 9 LPIYFDHRMH 1 is specified, the length of each peptide is 10 amino acids,the end position for each peptide is the start position plus nine

TABLE XXXVI 161P5C5 v.1: HLA Peptide Scoring Results A*0201 10-mersSYFPEITHI Pos 1234567890 score 31 IIFTNEFLTV 24 Portion 1 MLERGNVLSM 20of SEQ 22 YIRVRMFFSI 20 ID NO: 4; 2 LERGNVLSML 18 each 5 GNVLSMLIEV 17start 37 FLTVVNDKPI 17 position 61 MLAIYFDHRM 17 is 7 VLSMLIEVEF 15specified, 30 SIIFTNEFLT 15 the 32 IFTNEFLTVV 15 length 10 MLIEVEFRDR 14of each 26 RMFFSIIFTN 14 peptide 29 FSIIFTNEFL 13 is 10 9 SMLIEVEFRD 12amino 14 VEFRDRQAYI 12 acids, 55 QWFDANMLAI 12 the end 16 FRDRQAYIRV 11position 40 VVNDKPISFK 11 for each 53 NRQWFDANML 11 peptide 6 NVLSMLIEVE10 is the 23 IRVRMFFSII 10 start 33 FTNEFLTVVN 10 position 60 NMLAIYFDHR10 plus 11 LIEVEFRDRQ 9 nine 25 VRMFFSIIFT 9 58 DANMLAIYFD 9 12IEVEFRDRQA 8 8 LSMLIEVEFR 7 38 LTVVNDKPIS 7 41 VNDKPISFKA 7 50ACFNRQWFDA 7 52 FNRQWFDANM 7 62 LAIYFDHRMH 7 35 NEFLTVVNDK 6 39TVVNDKPISF 6 56 WFDANMLAIY 6 3 ERGNVLSMLI 5 18 DRQAYIRVRM 5 19RQAYIRVRMF 5 20 QAYIRVRMFF 5 17 RDRQAYIRVR 4 21 AYIRVRMFFS 4 24RVRMFFSIIF 4 28 FFSIIFTNEF 4 34 TNEFLTVVND 4 45 PISFKACFNR 4 46ISFKACFNRQ 4 49 KACFNRQWFD 4 54 RQWFDANMLA 4 44 KPISFKACFN 3 48FKACFNRQWF 3 57 FDANMLAIYF 3 59 ANMLAIYFDH 3 4 RGNVLSMLIE 2 51CFNRQWFDAN 2 27 MFFSIIFTNE 1 36 EFLTVVNDKP 1 42 NDKPISFKAC 1 43DKPISFKACF 1 47 SFKACFNRQW 1 15 EFRDRQAYIR −1 161P5C5 v.2: HLA PeptideScoring Results A*0201 10-mers SYFPEITHI Pos 1234567890 score 10SRQWFDANML 13 Portion of 7 ACFSRQWFDA 7 SEQ ID 9 FSRQWFDANM 7 NO: 6;each 2 PISFKACFSR 6 start 6 KACFSRQWFD 4 position is 1 KPISFKACFS 3specified, 3 ISFKACFSRQ 3 the length 5 FKACFSRQWF 3 of each 4 SFKACFSRQW1 peptide is 8 CFSRQWFDAN 1 10 amino acids, the end position for eachpeptide is the start position plus nine 161P5C5 v.3: HLA Peptide ScoringResults A*0201 10- mers SYFPEITHI Pos 1234567890 score 8 MLPIYFDHRM 15Portion of 7 NMLPIYFDHR 12 SEQ ID NO: 8; 2 QWFDANMLPI 11 each start 5DANMLPIYFD 9 position is 3 WFDANMLPIY 5 specified, 4 FDANMLPIYF 3 thelength 6 ANMLPIYFDH 3 of each 9 LPIYFDHRMH 3 peptide is 10 amino acids,the end position for each peptide is the start position plus nine

TABLE XXXVII 161P5C5 v.1 HLA Peptide Scoring Results A*0202 10- mersSYFPEITHI Pos 1234567890 score 19 RQAYIRVRMF 3 Portion of 48 FKACFNRQWF3 SEQ ID NO: 4; 57 FDANMLAIYF 3 each start 61 MLAIYFDHRM 3 position is20 QAYIRVRMFF 2 specified, 49 KACFNRQWFD 2 the length 58 DANMLAIYFD 2 ofeach 62 LAIYFDHRMH 2 peptide is 21 AYIRVRMFFS 1 10 amino 50 ACFNRQWFDA 1acids, the 59 ANMLAIYFDH 1 end position for each peptide is the startposition plus nine 161P5C5 v.2 HLA Peptide Scoring Results A*0202 10-mers SYFPEITHI Pos 1234567890 score 5 FKACFSRQWF 3 Portion of 6KACFSRQWFD 2 SEQ ID NO: 6; 7 ACFSRQWFDA 1 each start position isspecified, the length of each peptide is 10 amino acids, the endposition for each peptide is the start position plus nine 161P5C5 v.3HLA Peptide Scoring Results A*0202 10- mers SYFPEITHI Pos 1234567890score 4 FDANMLPIYF 3 Portion of 5 DANMLPIYFD 2 SEQ ID NO: 8; 6ANMLPIYFDH 1 each start position is specified, the length of eachpeptide is 10 amino acids, the end position for each peptide is thestart position plus nine 161P5C5 v.2 HLA Peptide Scoring Results A*020310- mers SYFPEITHI Pos 1234567890 score 7 AC FSRQWFDA 10 Portion of SEQ8 CF SRQWFDAN 9 ID NO: 6; each 9 FS RQWFDANM 8 start position isspecified, the length of each peptide is 10 amino acids, the endposition for each peptide is the start position plus nine

TABLE XXXVIII 161P5C5 v.1 HLA Peptide Scoring Results A*0203 10- mersSYFPEITHI Pos 1234567890 score 12 IE VEFRDRQA 10 Portion of 41 VNDKPISFKA 10 SEQ ID NO: 4; 50 AC FNRQWFDA 10 each start 54 RQ WFDANMLA 10position is 13 EV EFRDRQAY 9 specified, 42 ND KPISFKAC 9 the length of51 CF NRQWFDAN 9 each peptide 55 QW FDANMLAI 9 is 10 amino 14 VEFRDRQAYI 8 acids, the 43 DK PISFKACF 8 end position 52 FN RQWFDANM 8 foreach 56 WF DANMLAIY 8 peptide is the start position plus nine

TABLE XXXIX 161P5C5 v.1 HLA Peptide Scoring Results A3 10- mersSYFPEITHI Pos 1234567890 score 40 VV NDKPISFK 24 Portion of 24 RVRMFFSIIF 21 SEQ ID 1 ML ERGNVLSM 18 NO: 4; each 7 VL SMLIEVEF 18 start10 ML IEVEFRDR 18 position is 13 EV EFRDRQAY 18 specified, 39 TVVNDKPISF 18 the length 31 II FTNEFLTV 17 of each 6 NV LSMLIEVE 16peptide is 17 RD RQAYIRVR 15 10 amino 20 QA YIRVRMFF 13 acides, the 22YI RVRMFFSI 13 end 30 SI IFTNEFLT 12 position 45 PI SFKACFNR 12 for each61 ML AIYFDHRM 12 peptide is 15 EF RDRQAYIR 11 the start 35 NE FLTVVNDK11 position 56 WF DANMLAIY 11 plus nine 19 RQ AYIRVRMF 10 37 FL TVVNDKPI10 2 LE RGNVLSML 9 11 LI EVEFRDRQ 9 21 AY IRVRMFFS 9 33 FT NEFLTVVN 9 44KP ISFKACFN 9 57 FD ANMLAIYF 9 23 IR VRMFFSII 8 26 RM FFSIIFTN 8 32 IFTNEFLTVV 8 43 DK PISFKACF 8 59 AN MLAIYFDH 8 60 NM LAIYFDHR 8 62 LAIYFDHRMH 8 4 RG NVLSMLIE 7 18 DR QAYIRVRM 7 47 SF KACFNRQW 7 48 FKACFNRQWF 7 55 QW FDANMLAI 7 12 IE VEFRDRQA 6 14 VE FRDRQAYI 6 52 FNRQWFDANM 6 8 LS MLIEVEFR 5 28 FF SIIFTNEF 5 42 ND KPISFKAC 5 54 RQWFDANMLA 5 9 SM LIEVEFRD 4 29 FS IIFTNEFL 4 34 TN EFLTVVND 4 50 ACFNRQWFDA 4 51 CF NRQWFDAN 4 53 NR QWFDANML 4 16 FR DRQAYIRV 3 49 KACFNRQWFD 3 3 ER GNVLSMLI 2 36 EF LTVVNDKP 2 46 IS FKACFNRQ 2 5 GNVLSMLIEV 1 27 MF FSIIFTNE 1 161P5C5 v.2 HLA Peptide Scoring Results A310- mers SYFPEITHI Pos 1234567890 score 2 PI SFKACFSR 14 Portion of 1 KPISFKACFS 9 SEQ ID 4 SF KACFSRQW 7 NO: 6; each 5 FK ACFSRQWF 7 start 9 FSRQWFDANM 6 position 3 IS FKACFSRQ 5 is 10 SR QWFDANML 5 specified, 8 CFSRQWFDAN 4 the length 6 KA CFSRQWFD 3 of each 7 AC FSRQWFDA 3 peptide is10 amino acids, the end position for each peptide is the start positionplus nine 161P5C5 v.3 HLA Peptide Scoring Results A3 10- mers SYFPEITHIPos 1234567890 score 8 ML PIYFDHRM 11 Portion of 7 NM LPIYFDHR 9 SEQ ID3 WF DANMLPIY 8 NO: 8; each 6 AN MLPIYFDH 8 start 9 LP IYFDHRMH 8position 4 FD ANMLPIYF 7 is 2 QW FDANMLPI 6 specified, 1 RQ WFDANMLP 5the length of each peptide is 10 amino acids, the end position for eachpeptide is the start position plus nine

TABLE XL 161P5C5 v.1 HLA Peptide Scoring Results A26 10- mers Pos1234567890 score 13 EVEFRDRQAY 28 Portion of 56 WFDANMLAIY 22 SEQ ID 1MLERGNVLSM 21 NO: 4; each 39 TVVNDKPISF 21 start 7 VLSMLIEVEF 20position is 24 RVRMFFSIIF 20 specified, 43 DKPISFKACF 18 the length 61MLAIYFDHRM 18 of each 28 FFSIIFTNEF 17 peptide is 2 LERGNVLSML 16 10amino 10 MLIEVEFRDR 16 acids, the 18 DRQAYIRVRM 16 end 19 RQAYIRVRMF 16position 31 IIFTNEFLTV 16 for each 40 VVNDKPISFK 16 peptide is 6NVLSMLIEVE 15 the start 22 YIRVRMFFSI 15 position 27 MFFSIIFTNE 14 plusnine 15 EFRDRQAYIR 13 33 FTNEFLTVVN 13 36 EFLTVVNDKP 13 30 SIIFTNEFLT 1248 FKACFNRQWF 12 11 LIEVEFRDRQ 11 20 QAYIRVRMFF 11 38 LTVVNDKPIS 11 45PISFKACFNR 11 51 CFNRQWFDAN 11 52 FNRQWFDANM 11 57 FDANMLAIYF 11 29FSIIFTNEFL 10 32 IFTNEFLTVV 10 58 DANMLAIYFD 10 53 NRQWFDANML 9 37FLTVVNDKPI 8 42 NDKPISFKAC 8 46 ISFKACFNRQ 8 47 SFKACFNRQW 8 55QWFDANMLAI 8 3 ERGNVLSMLI 7 16 FRDRQAYIRV 6 21 AYIRVRMFFS 6 26RMFFSIIFTN 6 34 TNEFLTVVND 6 35 NEFLTVVNDK 6 5 GNVLSMLIEV 5 17RDRQAYIRVR 5 23 IRVRMFFSII 5 60 NMLAIYFDHR 5 4 RGNVLSMLIE 4 8 LSMLIEVEFR4 25 VRMFFSIIFT 4 59 ANMLAIYFDH 4 12 IEVEFRDRQA 3 14 VEFRDRQAYI 3 41VNDKPISFKA 3 44 KPISFKACFN 3 62 LAIYFDHRMH 3 50 ACFNRQWFDA 2 9SMLIEVEFRD 1 54 RQWFDANMLA 1 161P5C5 v.2 HLA Peptide Scoring Results A2610- mers SYFPEITHI Pos 1234567890 score 5 FKACFSRQWF 12 Portion of 2PISFKACFSR 11 SEQ ID NO: 6; 8 CFSRQWFDAN 11 each start 9 FSRQWFDANM 11position is 3 ISFKACFSRQ 9 specified, 10 SRQWFDANML 9 the length 4SFKACFSRQW 8 of each 1 KPISFKACFS 3 peptide is 7 ACFSRQWFDA 3 10 aminoacids, the end position for each peptide is the start position plus nine161P5C5 v.3 HLA Peptide Scoring Results A26 10- mers SYFPEITHI Pos1234567890 score 3 WFDANMLPIY 22 Portion of 8 MLPIYFDHRM 18 SEQ ID NO:8; 4 FDANMLPIYF 11 each start 5 DANMLPIYFD 10 position is 2 QWFDANMLPI 7specified, 7 NMLPIYFDHR 7 the length 6 ANMLPIYFDH 4 of each 9 LPIYFDHRMH3 peptide is 1 RQWFDANMLP 1 10 amino acids, the end position for eachpeptide is the start position plus nine

TABLE XLI 161P5C5 v.1 HLA Peptide Scoring Results B*0702 10-mersSYFPEITHI Pos 1234567890 score 2 LERGNVLSML 14 Portion of SEQ 44KPISFKACFN 12 ID NO: 4; each 29 FSIIFTNEFL 11 start position 7VLSMLIEVEF 10 is specified, 18 DRQAYIRVRM 10 the length of 31 IIFTNEFLTV10 each peptide 53 NRQWFDANML 10 is 10 amino 1 MLERGNVLSM 9 acids, theend 19 RQAYIRVRMF 9 position for 22 YIRVRMFFSI 9 each peptide 24RVRMFFSIIF 9 is the start 28 FFSIIFTNEF 9 position plus 32 IFTNEFLTVV 9nine 52 FNRQWFDANM 9 55 QWFDANMLAI 9 3 ERGNVLSMLI 8 20 QAYIRVRMFF 8 23IRVRMFFSII 8 25 VRMFFSIIFT 8 41 VNDKPISFKA 8 48 FKACFNRQWF 8 50ACFNRQWFDA 8 12 IEVEFRDRQA 7 14 VEFRDRQAYI 7 16 FRDRQAYIRV 7 37FLTVVNDKPI 7 43 DKPISFKACF 7 54 RQWFDANMLA 7 57 FDANMLAIYF 7 61MLAIYFDHRM 7 5 GNVLSMLIEV 6 30 SIIFTNEFLT 6 39 TVVNDKPISF 6 17RDRQAYIRVR 4 33 FTNEFLTVVN 4 4 RGNVLSMLIE 3 8 LSMLIEVEFR 3 13 EVEFRDRQAY3 15 EFRDRQAYIR 3 21 AYIRVRMFFS 3 34 TNEFLTVVND 3 42 NDKPISFKAC 3 46ISFKACFNRQ 3 56 WFDANMLAIY 3 59 ANMLAIYFDH 3 27 MFFSIIFTNE 2 45PISFKACFNR 2 49 KACFNRQWFD 2 51 CFNRQWFDAN 2 60 NMLAIYFDHR 2 6NVLSMLIEVE 1 10 MLIEVEFRDR 1 11 LIEVEFRDRQ 1 26 RMFFSIIFTN 1 35NEFLTVVNDK 1 36 EFLTVVNDKP 1 38 LTVVNDKPIS 1 40 VVNDKPISFK 1 47SFKACFNRQW 1 58 DANMLAIYFD 1 161P5C5 v.2 HLA Peptide Scoring ResultsB*0702 10-mers SYFPEITHI Pos 1234567890 score 1 KPISFKACFS 12 10SRQWFDANML 10 9 FSRQWFDANM 9 5 FKACFSRQWF 8 7 ACFSRQWFDA 8 8 CFSRQWFDAN4 3 ISFKACFSRQ 3 2 PISFKACFSR 2 6 KACFSRQWFD 2 4 SFKACFSRQW 1 161P5C5v.3 HLA Peptide Scoring Results B*0702 10-mers SYFPEITHI Pos 1234567890score 9 LPIYFDHRMH 10 Portion of 2 QWFDANMLPI 9 SEQ ID NO: 8; 4FDANMLPIYF 7 each start 8 MLPIYFDHRM 6 position is 3 WFDANMLPIY 3specified, 6 ANMLPIYFDH 3 the length 7 NMLPIYFDHR 2 of each 1 RQWFDANMLP1 peptide is 5 DANMLPIYFD <tda 10 amino acids, the end position for eachpeptide is the start position plus nine

TABLE XLII 161P5C5: HLA Peptide Scoring Results B*08 10-mers SYFPEITHIPos 1234567890 NO DATA

TABLE XLIII 161P5C5: HLA Peptide Scoring Results B*1510 10-mersSYFPEITHI Pos 1234567890 NO DATA

TABLE XLIV 161P5C5: HLA Peptide Scoring Results B*2705 10-mers SYFPEITHIPos 1234567890 NO DATA

TABLE XLV 161P5C5: HLA Peptide Scoring Results B*2709 10-mers SYFPEITHIPos 1234567890 NO DATA

TABLE XLVI 161P5C5 v.1 HLA Peptide Scoring Results B*4402 10-mersSYFPEITHI Pos 1234567890 score 2 LERGNVLSML 22 Portion of 14 VEFRDRQAYI21 SEQ ID 35 NEFLTVVNDK 16 NO: 4; each 13 EVEFRDRQAY 15 start 55QWFDANMLAI 14 position 28 FFSIIFTNEF 13 is 29 FSIIFTNEFL 13 specified,39 TVVNDKPISF 13 the length 47 SFKACFNRQW 13 of each 53 NRQWFDANML 13peptide is 57 FDANMLAIYF 13 10 amino 3 ERGNVLSMLI 12 acids, the 7VLSMLIEVEF 12 end 12 IEVEFRDRQA 12 position 19 RQAYIRVRMF 12 for each 56WFDANMLAIY 12 peptide is 20 QAYIRVRMFF 11 the start 24 RVRMFFSIIF 11position 43 DKPISFKACF 11 plus nine 48 FKACFNRQWF 11 23 IRVRMFFSII 10 37FLTVVNDKPI 10 22 YIRVRMFFSI 9 42 NDKPISFKAC 9 21 AYIRVRMFFS 7 6NVLSMLIEVE 6 26 RMFFSIIFTN 6 31 IIFTNEFLTV 6 50 ACFNRQWFDA 6 10MLIEVEFRDR 5 17 RDRQAYIRVR 5 25 VRMFFSIIFT 5 30 SIIFTNEFLT 5 36EFLTVVNDKP 5 44 KPISFKACFN 5 59 ANMLAIYFDH 5 15 EFRDRQAYIR 4 40VVNDKPISFK 4 46 ISFKACFNRQ 4 51 CFNRQ&n

TABLE XLVII 161P5C5: HLA Peptide Scoring Results B*5101 10-mersSYFPEITHI Pos 1234567890 NO DATA

TABLE XLVIII 161P5C5 v.1 HLA Peptide Scoring Results DRB1*0101 15- mersSYFPEITHI Pos 123456789012345 score 26 RMFFSIIFTNEFLTV 33 Portion of SEQID NO: 4; each 5 GNVLSMLIEVEFRDR 25 start position is specified, the 22YIRVRMFFSIIFTNE 25 length of each peptide is 15 34 TNEFLTVVNDKPISF 25amino acids, the end position for 19 RQAYIRVRMFFSIIF 24 each peptide isthe start 35 NEFLTVVNDKPISFK 24 position plus fourteen 37FLTVVNDKPISFKAC 23 55 QWFDANMLAIYFDHR 22 1 MLERGNVLSMLIEVE 20 13EVEFRDRQAYIRVRM 20 30 SIIFTNEFLTVVNDK 20 20 QAYIRVRMFFSIIFT 18 53NRQWFDANMLAIYFD 18 25 VRMFFSIIFTNEFLT 17 29 FSIIFTNEFLTVVND 17 39TVVNDKPISFKACFN 17 49 KACFNRQWFDANMLA 17 8 LSMLIEVEFRDRQAY 16 51CFNRQWFDANMLAIY 16 17 RDRQAYIRVRMFFSI 15 21 AYIRVRMFFSIIFTN 15 4RGNVLSMLIEVEFRD 14 14 VEFRDRQAYIRVRMF 14 45 PISFKACFNRQWFDA 12 7VLSMLIEVEFRDRQA 11 18 DRQAYIRVRMFFSII 11 27 MFFSIIFTNEFLTVV 11 41VNDKPISFKACFNRQ 11 3 ERGNVLSMLIEVEFR 10 10 MLIEVEFRDRQAYIR 10 50ACFNRQWFDANMLAI 10 54 RQWFDANMLAIYFDH 10 56 WFDANMLAIYFDHRM 10 2LERGNVLSMLIEVEF 9 11 LIEVEFRDRQAYIRV 9 24 RVRMFFSIIFTNEFL 9 28FFSIIFTNEFLTVVN 9 32 IFTNEFLTVVNDKPI 9 36 EFLTVVNDKPISFKA 9 38LTVVNDKPISFKACF 9 43 DKPISFKACFNRQWF 9 52 FNRQWFDANMLAIYF 9 6NVLSMLIEVEFRDRQ 8 9 SMLIEVEFRDRQAYI 8 12 IEVEFRDRQAYIRVR 8 16FRDRQAYIRVRMFFS 8 40 VVNDKPISFKACFNR 8 42 NDKPISFKACFNRQW 8 46ISFKACFNRQWFDAN 8 48 FKACFNRQWFDANML 8 31 IIFTNEFLTVVNDKP 7 15EFRDRQAYIRVRMFF 3 44 KPISFKACFNRQWFD 2 23 IRVRMFFSIIFTNEF 1 33FTNEFLTVVNDKPIS 1 47 SFKACFNRQWFDANM 1 57 FDANMLAIYFDHRMH 1 161P5C5 v.2HLA Peptide Scoring Results DRB1*0101 15- mers SYFPEITHI Pos123456789012345 score 7 PISFKACFSRQWFDA 18 Portion of SEQ ID NO: 6; each15 SRQWFDANMLAIYFD 18 start position is specified, the 1 TVVNDKPISFKACFS17 length of each peptide is 15 4 NDKPISFKACFSRQW 16 amino acids, theend position for 11 KACFSRQWFDANMLA 16 each peptide is the start 13CFSRQWFDANMLAIY 16 position plus fourteen 3 VNDKPISFKACFSRQ 11 12ACFSRQWFDANMLAI 10 5 DKPISFKACFSRQWF 9 14 FSRQWFDANMLAIYF 9 2VVNDKPISFKACFSR 8 8 ISFKACFSRQWFDAN 8 10 FKACFSRQWFDANML 8 6KPISFKACFSRQWFD 2 9 SFKACFSRQWFDANM 1 161P5C5 v.3 HLA Peptide ScoringResults DRB1*0101 15- mers SYFPEITHI Pos 123456789012345 score 7QWFDANMLPIYFDHR 20 Portion of SEQ ID NO: 8; each 5 NRQWFDANMLPIYFD 18start position is specified, the 1 KACFNRQWFDANMLP 17 length of eachpeptide is 15 3 CFNRQWFDANMLPIY 16 amino acids, the end position for 2ACFNRQWFDANMLPI 10 each peptide is the start 6 RQWFDANMLPIYFDH 10position plus fourteen 8 WFDANMLPIYFDHRM 10 4 FNRQWFDANMLPIYF 9 9FDANMLPIYFDHRMH 1

TABLE XLIX 161P5C5 v.1 HLA Peptide Scoring Results DRB1*0301 15- mersSYFPEITHI Pos 123456789012345 score 37 FLTVVNDKPISFKAC 30 Portion of SEQID NO: 4; each start 5 GNVLSMLIEVEFRDR 26 position is specified, thelength 11 LIEVEFRDRQAYIRV 26 of each peptide is 15 amino acids, 22YIRVRMFFSIIFTNE 20 the end position for each peptide 7 VLSMLIEVEFRDRQA18 is the start position plus fourteen 28 FFSIIFTNEFLTVVN 18 12IEVEFRDRQAYIRVR 17 26 RMFFSIIFTNEFLTV 17 34 TNEFLTVVNDKPISF 17 52FNRQWFDANMLAIYF 17 54 RQWFDANMLAIYFDH 17 9 SMLIEVEFRDRQAYI 16 27MFFSIIFTNEFLTVV 15 41 VNDKPISFKACFNRQ 15 45 PISFKACFNRQWFDA 15 46ISFKACFNRQWFDAN 15 4 RGNVLSMLIEVEFRD 14 18 DRQAYIRVRMFFSII 14 8LSMLIEVEFRDRQAY 13 29 FSIIFTNEFLTVVND 12 20 QAYIRVRMFFSIIFT 11 35NEFLTVVNDKPISFK 11 38 LTVVNDKPISFKACF 11 43 DKPISFKACFNRQWF 11 24RVRMFFSIIFTNEFL 10 25 VRMFFSIIFTNEFLT 10 13 EVEFRDRQAYIRVRM 9 30SIIFTNEFLTVVNDK 9 49 KACFNRQWFDANMLA 9 51 CFNRQWFDANMLAIY 9 55QWFDANMLAIYFDHR 9 17 RDRQAYIRVRMFFSI 8 16 FRDRQAYIRVRMFFS 7 36EFLTVVNDKPISFKA 7 47 SFKACFNRQWFDANM 6 40 VVNDKPISFKACFNR 4 31IIFTNEFLTVVNDKP 3 42 NDKPISFKACFNRQW 3 50 ACFNRQWFDANMLAI 3 1MLERGNVLSMLIEVE 2 2 LERGNVLSMLIEVEF 2 3 ERGNVLSMLIEVEFR 2 6NVLSMLIEVEFRDRQ 2 10 MLIEVEFRDRQAYIR 2 32 IFTNEFLTVVNDKPI 2 33FTNEFLTVVNDKPIS 2 53 NRQWFDANMLAIYFD 2 15 EFRDRQAYIRVRMFF 1 19RQAYIRVRMFFSIIF 1 21 AYIRVRMFFSIIFTN 1 23 IRVRMFFSIIFTNEF 1 39TVVNDKPISFKACFN 1 44 KPISFKACFNRQWFD 1 56 WFDANMLAIYFDHRM 1 57FDANMLAIYFDHRMH 1 161P5C5 v.2 HLA Peptide Scoring Results DRB1*0301 15-mers SYFPEITHI Pos 123456789012345 score 14 FSRQWFDANMLAIYF 17 Portionof SEQ ID NO: 6; each 8 ISFKACFSRQWFDAN 16 start position is specified,the 3 VNDKPISFKACFSRQ 15 length of each peptide is 15 5 DKPISFKACFSRQWF10 amino acids, the end position for 7 PISFKACFSRQWFDA 9 each peptide isthe start 11 KACFSRQWFDANMLA 9 position plus fourteen 13 CFSRQWFDANMLAIY9 9 SFKACFSRQWFDANM 6 2 VVNDKPISFKACFSR 4 4 NDKPISFKACFSRQW 3 12ACFSRQWFDANMLAI 3 15 SRQWFDANMLAIYFD 2 1 TVVNDKPISFKACFS 1 10FKACFSRQWFDANML 1 161P5C5 v.3 HLA Peptide Scoring Results DRB1*0301 15-mers SYFPEITHI Pos 123456789012345 score 4 FNRQWFDANMLPIYF 17 Portion ofSEQ ID NO: 8; each 6 RQWFDANMLPIYFDH 16 start position is specified, the1 KACFNRQWFDANMLP 9 length of each peptide is 15 3 CFNRQWFDANMLPIY 9amino acids, the end position for 7 QWFDANMLPIYFDHR 9 each peptide isthe start 2 ACFNRQWFDANMLPI 3 position plus fourteen 8 WFDANMLPIYFDHRM 25 NRQWFDANMLPIYFD 1 9 FDANMLPIYFDHRMH 1

TABLE L 161P5C5 v.1 HLA Peptide Scoring Results DRB1*0401 15-mersSYFPEITHI Pos 123456789012345 score 9 SMLIEVEFRDRQAYI 26 Portion of 19RQAYIRVRMFFSIIF 22 SEQ ID 26 RMFFSIIFTNEFLTV 22 NO: 4; each 30SIIFTNEFLTVVNDK 22 start 34 TNEFLTVVNDKPISF 22 position is 53NRQWFDANMLAIYFD 22 specified, 22 YIRVRMFFSIIFTNE 20 the length 35NEFLTVVNDKPISFK 20 of each 1 MLERGNVLSMLIEVE 18 peptide is 12IEVEFRDRQAYIRVR 18 15 amino 27 MFFSIIFTNEFLTVV 18 acids, the 31IIFTNEFLTVVNDKP 18 end 52 FNRQWFDANMLAIYF 18 position 25 VRMFFSIIFTNEFLT16 for each 45 PISFKACFNRQWFDA 16 peptide is 49 KACFNRQWFDANMLA 16 thestart 5 GNVLSMLIEVEFRDR 14 position 7 VLSMLIEVEFRDRQA 14 plus 8LSMLIEVEFRDRQAY 14 fourteen 24 RVRMFFSIIFTNEFL 14 28 FFSIIFTNEFLTVVN 1429 FSIIFTNEFLTVVND 14 37 FLTVVNDKPISFKAC 14 10 MLIEVEFRDRQAYIR 12 16FRDRQAYIRVRMFFS 12 23 IRVRMFFSIIFTNEF 12 39 TVVNDKPISFKACFN 12 42NDKPISFKACFNRQW 12 44 KPISFKACFNRQWFD 12 46 ISFKACFNRQWFDAN 12 51CFNRQWFDANMLAIY 12 54 RQWFDANMLAIYFDH 10 11 LIEVEFRDRQAYIRV 9 20QAYIRVRMFFSIIFT 9 38 LTVVNDKPISFKACF 9 4 RGNVLSMLIEVEFRD 8 2LERGNVLSMLIEVEF 6 3 ERGNVLSMLIEVEFR 6 6 NVLSMLIEVEFRDRQ 6 14VEFRDRQAYIRVRMF 6 15 EFRDRQAYIRVRMFF 6 17 RDRQAYIRVRMFFSI 6 21AYIRVRMFFSIIFTN 6 32 IFTNEFLTVVNDKPI 6 33 FTNEFLTVVNDKPIS 6 36EFLTVVNDKPISFKA 6 40 VVNDKPISFKACFNR 6 47 SFKACFNRQWFDANM 6 50ACFNRQWFDANMLAI 6 56 WFDANMLAIYFDHRM 6 57 FDANMLAIYFDHRMH 6 13EVEFRDRQAYIRVRM 5 43 DKPISFKACFNRQWF 3 18 DRQAYIRVRMFFSII 1 48FKACFNRQWFDANML 1 161P5C5 v.2 HLA Peptide Scoring Results DRB1*040115-mers SYFPEITHI Pos 123456789012345 score 15 SRQWFDANMLAIYFD 22Portion of SEQ ID NO: 6; each 14 FSRQWFDANMLAIYF 18 start position isspecified, 7 PISFKACFSRQWFDA 16 the length of each peptide is 11KACFSRQWFDANMLA 16 15 amino acids, the end 1 TVVNDKPISFKACFS 12 positionfor each peptide is 4 NDKPISFKACFSRQW 12 the start position plus 6KPISFKACFSRQWFD 12 fourteen 8 ISFKACFSRQWFDAN 12 13 CFSRQWFDANMLAIY 12 2VVNDKPISFKACFSR 6 9 SFKACFSRQWFDANM 6 12 ACFSRQWFDANMLAI 6 5DKPISFKACFSRQWF 3 10 FKACFSRQWFDANML 1 161P5C5 v.3 HLA Peptide ScoringResults DRB1*0401 15-mers SYFPEITHI Pos 123456789012345 score 5NRQWFDANMLPIYFD 22 Portion of SEQ ID NO: 8; each start 1 KACFNRQWFDANMLP16 position is specified, the length 3 CFNRQWFDANMLPIY 12 of eachpeptide is 15 amino acids, 4 FNRQWFDANMLPIYF 12 the end position foreach peptide 6 RQWFDANMLPIYFDH 10 is the start position plus 2ACFNRQWFDANMLPI 6 fourteen 8 WFDANMLPIYFDHRM 6

TABLE LI 161P5C5 v.1 HLA Peptide Scoring Results DRB1*1101 15- mersSYFPEITHI Pos 123456789012345 score 34 TNEFLTVVNDKPISF 25 Portion of SEQID NO: 19 RQAYIRVRMFFSIIF 22 4; each start position 9 SMLIEVEFRDRQAYI 17is specified, the 25 VRMFFSIIFTNEFLT 17 length of each peptide 26RMFFSIIFTNEFLTV 16 is 15 amino acids, the 36 EFLTVVNDKPISFKA 16 endposition for each 35 NEFLTVVNDKPISFK 15 peptide is the start 11LIEVEFRDRQAYIRV 14 position plus fourteen 4 RGNVLSMLIEVEFRD 13 8LSMLIEVEFRDRQAY 13 30 SIIFTNEFLTVVNDK 13 5 GNVLSMLIEVEFRDR 12 13EVEFRDRQAYIRVRM 11 45 PISFKACFNRQWFDA 11 53 NRQWFDANMLAIYFD 11 54RQWFDANMLAIYFDH 11 7 VLSMLIEVEFRDRQA 10 16 FRDRQAYIRVRMFFS 10 18DRQAYIRVRMFFSII 10 49 KACFNRQWFDANMLA 10 2 LERGNVLSMLIEVEF 8 41VNDKPISFKACFNRQ 8 46 ISFKACFNRQWFDAN 8 1 MLERGNVLSMLIEVE 7 10MLIEVEFRDRQAYIR 7 17 RDRQAYIRVRMFFSI 7 20 QAYIRVRMFFSIIFT 7 21AYIRVRMFFSIIFTN 7 22 YIRVRMFFSIIFTNE 7 24 RVRMFFSIIFTNEFL 7 28FFSIIFTNEFLTVVN 7 29 FSIIFTNEFLTVVND 7 39 TVVNDKPISFKACFN 7 48FKACFNRQWFDANML 7 52 FNRQWFDANMLAIYF 7 6 NVLSMLIEVEFRDRQ 6 32IFTNEFLTVVNDKPI 6 37 FLTVVNDKPISFKAC 6 38 LTVVNDKPISFKACF 6 40VVNDKPISFKACFNR 6 43 DKPISFKACFNRQWF 6 55 QWFDANMLAIYFDHR 6 56WFDANMLAIYFDHR&

TABLE LII Peptides Used to Generate HLA Tables and Scoring Results andPosition Determination Key 161P5C5 v.1 aa 1-71 nonamers decamers and15-mers (SEQ ID NO: 43) MLERGNVLSM LIEVEFRDRQ AYIRVRMFFS IIFTNEFLTVVNDKPISFKA CFNRQWFDAN MLAIYFDHRM H 161P5C5 v.2 nonamers aa45-61 (SEQ IDNO: 44) PISFKACFSRQWFDANM decamers aa44-62 (SEQ ID NO: 45)KPISFKACFSRQWFDANML 15-mers aa39-67 (SEQ ID NO: 46)TVVNDKPISFKACFSRQWFDANMLAIYFD 161P5C5 v.3 Nonamers aa 55-71 (SEQ ID NO:47) QWFDANMLPIYFDHRMH Decamers aa 54-71 (SEQ ID NO: 48)RQWFDANMLPIYFDHRMH 15-mers aa 49-71 (SEQ ID NO: 49)KACFNRQWFDANMLPIYFDHRMH

TABLE LIII Exon compositions of 161P5C5 v.1 Exon Number Start End Exon 11 297 Exon 2 298 436 Exon 3 437 1568

TABLE LIV Nucleotide sequence of transcript variant 161P5C5 v.7 (SEQ IDNO: 50) attaaattaa cgggatatac tttataaata atacttaggg aagatgatatagttagagga 60 ccaggattct aggtctagcc actaactagc tgtgtgaact tagacaaatcatttaatttt 120 atttaattca gaaaaatata tgaaaacaaa gtagctcaat ctcactgctcacataactac 180 ttgtaaagaa atacatgtac agagtttgtt cttctttttt atcatttcaggaaatgaata 240 acatgaagtg aagtcttcat ctccattccc aacagtcccc attctacttgcagaaaggtt 300 gcttacactg aaaatcagtt tattttcccc tggtgcaaag aacagtcgtttctccaaaac 360 tgaagctgga aattatctga aatatcaggt cctccggaaa agggacgtgaagcccccttt 420 gtaatttctg cattagcgtg ctctcctggc aagcaggaaa cctcatcagagaagtcagcc 480 aaggaaagtc tttaaatgga aattgtgcaa acgaggagca aatgcattaaaaagttgctg 540 acgggcatga aatgctttga tgtgaagacg gaaaactcca agcaggaaggattttaacat 600 tttgaatctg attgactctg tggtttctca gcacagttat tccatgggctaaaataaatg 660 cagaaatggt actttcagac cacagctgca gaggggatcg tggtgaatttcaatgaaaat 720 ccatttgaat cttgaggttc agatcttaaa aaagcaaagg acatgagagaagtaatattg 780 ttgcttgaaa tttcattgct tatatctaaa agaaactcct atttttaagagaaatgttga 840 atctttgcaa cgtggtagac gctcccacaa aactttctcc tgaaataggagataaatgtt 900 ggaaagaggc aatgtattga gtatgctgat agaggtggag ttcagagacaggcaagcata 960 cataagagtc aggatgtttt tcagtattat ctttacaaat gagtttcttacagtggtcaa 1020 tgacaaacca atttcattca aagcttgctt caataggcaa tggtttgatgccaatatgtt 1080 agctatttac tttgaccacc gtatgcatta aaaagaagaa aaattaagaatactcaagca 1140 gaacctccaa cttagatagc actttccaca aaaagtaatg gagggatagactgaagttaa 1200 atgggatcag gtatgtgatg agatctcaga agtgtttgca caataatgcagatactcatt 1260 ttaaacagag tcataaggat tggaactaat aaaaataata gaataaaataccgatcaaga 1320 atgtgaaaaa aacctcctgc gtatctgggt tttgaattct ggctccacagaacttgtcag 1380 atatatgaca ttaaacagag atacttcaaa aaaaaaaaaa aaaaaaaaa1429

TABLE LV Nucleotide sequence alignment of 121P1F1 v.1 (SEQ ID NO: 51)and 161P5C5 v.7 (SEQ ID NO: 52). 161P5C5v.1ATTAAATTAACGGGATATACTTTATAAATAATACTTAGGGAAGATGATATAGTTAGAGGA 60161P5C5v.7 ATTAAATTAACGGGATATACTTTATAAATAATACTTAGGGAAGATGATATAGTTAGAGGA60 ************************************************************161P5C5v.1 CCAGGATTCTAGGTCTAGCCACTAACTAGCTGTGTGAACTTAGACAAATCATTTAATTTT120 161P5C5v.7CCAGGATTCTAGGTCTAGCCACTAACTAGCTGTGTGAACTTAGACAAATCATTTAATTTT 120************************************************************ 161P5C5v.1ATTTAATTCAGAAAAATATATGAAAACAAAGTAGCTCAATCTCACTGCTCACATAACTAC 180161P5C5v.7 ATTTAATTCAGAAAAATATATGAAAACAAAGTAGCTCAATCTCACTGCTCACATAACTAC180 ************************************************************161P5C5v.1 TTGTAAAGAAATACATGTACAGAGTTTGTTCTTCTTTTTTATCATTTCAGGAAATGAATA240 161P5C5v.7TTGTAAAGAAATACATGTACAGAGTTTGTTCTTCTTTTTTATCATTTCAGGAAATGAATA 240************************************************************ 161P5C5v.1ACATGAAGTGAAGTCTTCATCTCCATTCCCAACAGTCCCCATTCTACTTGCAGAAAGCTT 300161P5C5v.7 ACATGAAGTGAAGTCTTCATCTCCATTCCCAACAGTCCCCATTCTACTTGCAGAAAG---297 ********************************************************* 161P5C5v.1TTCCAAAACTCTTTAATGAAAAGTCAGCAAGAAGATAATGAGAAAGGACCAAACAGATGT 360161P5C5v.7 ------------------------------------------------------------161P5C5v.1 TGGCTTCTGCTGAAATTTGCCAAACTTTTACAGCATCATTATGATAGCTTTCCGTTTAGG420 161P5C5v.7------------------------------------------------------------ 161P5C5v.1TCACCACAGTTTAAAAGTTGCTTACACTGAAAATCAGTTTATTTTCCCCTGGTGCAAAGA 480161P5C5v.7 ----------------GTTGCTTACACTGAAAATCAGTTTATTTTCCCCTGGTGCAAAGA341                 ********************************************161P5C5v.1 ACAGTCGTTTCTCCAAAACTGAAGCTGGAAATTATCTGAAATATCAGGTCCTCCGGAAAA540 161P5C5v.7ACAGTCGTTTCTCCAAAACTGAAGCTGGAAATTATCTGAAATATCAGGTCCTCCGGAAAA 401************************************************************ 161P5C5v.1GGGACGTGAAGCCCCCTTTGTAATTTCTGCATTAGCGTGCTCTCCTGGCAAGCAGGAAAC 600161P5C5v.7 GGGACGTGAAGCCCCCTTTGTAATTTCTGCATTAGCGTGCTCTCCTGGCAAGCAGGAAAC461 ************************************************************161P5C5v.1 CTCATCAGAGAAGTCAGCCAAGGAAAGTCTTTAAATGGAAATTGTGCAAACGAGGAGCAA660 161P5C5v.7CTCATCAGAGAAGTCAGCCAAGGAAAGTCTTTAAATGGAAATTGTGCAAACGAGGAGCAA 521************************************************************ 161P5C5v.1ATGCATTAAAAAGTTGCTGACGGGCATGAAATGCTTTGATGTGAAGACGGAAAACTCCAA 720161P5C5v.7 ATGCATTAAAAAGTTGCTGACGGGCATGAAATGCTTTGATGTGAAGACGGAAAACTCCAA581 ************************************************************161P5C5v.1 GCAGGAAGGATTTTAACATTTTGAATCTGATTGACTCTGTGGTTTCTCAGCACAGTTATT780 161P5C5v.7GCAGGAAGGATTTTAACATTTTGAATCTGATTGACTCTGTGGTTTCTCAGCACAGTTATT 641************************************************************ 161P5C5v.1CCATGGGCTAAAATAAATGCAGAAATGGTACTTTCAGACCACAGCTGCAGAGGGGATCGT 840161P5C5v.7 CCATGGGCTAAAATAAATGCAGAAATGGTACTTTCAGACCACAGCTGCAGAGGGGATCGT701 ************************************************************161P5C5v.1 GGTGAATTTCAATGAAAATCCATTTGAATCTTGAGGTTCAGATCTTAAAAAAGCAAAGGA900 161P5C5v.7GGTGAATTTCAATGAAAATCCATTTGAATCTTGAGGTTCAGATCTTAAAAAAGCAAAGGA 761************************************************************ 161P5C5v.1CATGAGAGAAGTAATATTGTTGCTTGAAATTTCATTGCTTATATCTAAAAGAAACTCCTA 960161P5C5v.7 CATGAGAGAAGTAATATTGTTGCTTGAAATTTCATTGCTTATATCTAAAAGAAACTCCTA821 ************************************************************161P5C5v.1 TTTTTAAGAGAAATGTTGAATCTTTGCAACGTGGTAGACGCTCCCACAAAACTTTCTCCT1020 161P5C5v.7TTTTTAAGAGAAATGTTGAATCTTTGCAACGTGGTAGACGCTCCCACAAAACTTTCTCCT 881************************************************************ 161P5C5v.1GAAATAGGAGATAAATGTTGGAAAGAGGCAATGTATTGAGTATGCTGATAGAGGTGGAGT 1080161P5C5v.7 GAAATAGGAGATAAATGTTGGAAAGAGGCAATGTATTGAGTATGCTGATAGAGGTGGAGT941 ************************************************************161P5C5v.1 TCAGAGACAGGCAAGCATACATAAGAGTCAGGATGTTTTTCAGTATTATCTTTACAAATG1140 161P5C5v.7TCAGAGACAGGCAAGCATACATAAGAGTCAGGATGTTTTTCAGTATTATCTTTACAAATG 1001************************************************************ 161P5C5v.1AGTTTCTTACAGTGGTCAATGACAAACCAATTTCATTCAAAGCTTGCTTCAATAGGCAAT 1200161P5C5v.7 AGTTTCTTACAGTGGTCAATGACAAACCAATTTCATTCAAAGCTTGCTTCAATAGGCAAT1061 ************************************************************161P5C5v.1 GGTTTGATGCCAATATGTTAGCTATTTACTTTGACCACCGTATGCATTAAAAAGAAGAAA1260 161P5C5v.7GGTTTGATGCCAATATGTTAGCTATTTACTTTGACCACCGTATGCATTAAAAAGAAGAAA 1121************************************************************ 161P5C5v.1AATTAAGAATACTCAAGCAGAACCTCCAACTTAGATAGCACTTTCCACAAAAAGTAATGG 1320161P5C5v.7 AATTAAGAATACTCAAGCAGAACCTCCAACTTAGATAGCACTTTCCACAAAAAGTAATGG1181 ************************************************************161P5C5v.1 AGGGATAGACTGAAGTTAAATGGGATCAGGTATGTGATGAGATCTCAGAAGTGTTTGCAC1380 161P5C5v.7AGGGATAGACTGAAGTTAAATGGGATCAGGTATGTGATGAGATCTCAGAAGTGTTTGCAC 1241************************************************************ 161P5C5v.1AATAATGCAGATACTCATTTTAAACAGAGTCATAAGGATTGGAACTAATAAAAATAATAG 1440161P5C5v.7 AATAATGCAGATACTCATTTTAAACAGAGTCATAAGGATTGGAACTAATAAAAATAATAG1301 ************************************************************161P5C5v.1 AATAAAATACCGATCAAGAATGTGAAAAAAACCTCCTGCGTATCTGGGTTTTGAATTCTG1500 161P5C5v.7AATAAAATACCGATCAAGAATGTGAAAAAAACCTCCTGCGTATCTGGGTTTTGAATTCTG 1361************************************************************ 161P5C5v.1GCTCCACAGAACTTGTCAGATATATGACATTAAACAGAGATACTTCAAAAAAAAAAAAAA 1560161P5C5v.7 GCTCCACAGAACTTGTCAGATATATGACATTAAACAGAGATACTTCAAAAAAAAAAAAAA1421 ************************************************************161P5C5v.1 AAAAAAAA 1568 161P5C5v.7 AAAAAAAA 1429 ********

TABLE LVI Peptide sequences of protein coded by 161P5C5 v.7 (SEQ ID NO:53) MLERGNVLSM LIEVEFRDRQ AYIRVRMFFS IIFTNEFLTV 60 VNDKPISFKA CFNRQWFDANMLAIYFDHRM H 71

TABLE LVII Amino acid sequence alignment of 121P1F1 v.1 (SEQ ID NO: 54)and 161P5C5 v.7 (SEQ ID NO: 55) 161P5C5v.1MLERGNVLSMLIEVEFRDRQAYIRVRMFFSIIFTNEFLTVVNDKPISFKACFNRQWFDAN 60161P5C5v.7 MLERGNVLSMLIEVEFRDRQAYIRVRMFFSIIFTNEFLTVVNDKPISFKACFNRQWFDAN60 ************************************************************161P5C5v.1 MLAIYFDHRMH 71 161P5C5v.7 MLAIYFDHRMH 71 ***********

1. An isolated protein, comprising the polypeptide sequence of SEQ IDNO: 4, SEQ ID NO: 6, or SEQ ID NO:
 8. 2. A composition, comprising: aphysiologically acceptable carrier; and a protein comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:4, SEQ IDNO:6, and SEQ ID NO:8.
 3. The composition of claim 2, wherein theprotein comprises the amino acid sequence of SEQ ID NO:
 4. 4. Thecomposition of claim 2, further comprising an adjuvant.
 5. Thecomposition of claim 2, wherein the protein further comprises aheterologous polypeptide.
 6. The isolated protein of claim 1, furthercomprising a heterologous polypeptide.
 7. An isolated protein,comprising the polypeptide sequence of SEQ ID NO:
 4. 8. The isolatedprotein of claim 7, further comprising a heterologous polypeptide.
 9. Acomposition, comprising the protein of claim 7 and a physiologicallyacceptable carrier.
 10. The composition of claim 9, further comprisingan adjuvant.